CN102171349A - Method for producing chemical and continuous culture system - Google Patents

Method for producing chemical and continuous culture system Download PDF

Info

Publication number
CN102171349A
CN102171349A CN2009801386054A CN200980138605A CN102171349A CN 102171349 A CN102171349 A CN 102171349A CN 2009801386054 A CN2009801386054 A CN 2009801386054A CN 200980138605 A CN200980138605 A CN 200980138605A CN 102171349 A CN102171349 A CN 102171349A
Authority
CN
China
Prior art keywords
nutrient solution
membrane sepn
membrane
culture tank
sepn groove
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN2009801386054A
Other languages
Chinese (zh)
Inventor
耳冢孝
石井健太郎
守田健
日笠雅史
泽井健司
泽井秀树
山田胜成
峰岸进一
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toray Industries Inc
Original Assignee
Toray Industries Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toray Industries Inc filed Critical Toray Industries Inc
Priority to CN201610340983.2A priority Critical patent/CN106011187A/en
Publication of CN102171349A publication Critical patent/CN102171349A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Sustainable Development (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Water Supply & Treatment (AREA)
  • Cell Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

To control the flow rate of a liquid culture medium in a membrane separation tank without affecting the culture conditions in a culture tank and to increase the production efficiency of a chemical by inhibiting the precipitation of a microorganism or culture cells, provided is a method for producing a chemical which comprises culturing a microorganism or culture cells in a culture tank, feeding the liquid culture medium from the culture tank to a membrane separation tank and filtering the same through a separation membrane, collecting a fermentation product as the chemical from the permeate and, at the same time, returning the unfiltered liquid culture medium not having been filtered in such a manner as combining the same with the liquid culture medium in the upstream side of the membrane separation tank, wherein a portion of the liquid culture medium fed from the culture tank is passed outside the membrane separation tank depending on the inflow side pressure of the membrane separation tank.

Description

The manufacture method of chemical and bactogen
Technical field
The present invention relates to method by the cultivation manufacturing chemistry product of microorganism or culturing cell.Relate to make with high productivity the manufacture method and the culture apparatus of the chemical of desired chemical in more detail, this manufacture method is by cultivating on one side, on one side from the nutrient solution of microorganism or culturing cell, effectively filter the liquid that contains the fermentative production thing (chemical) that cultivation produces, reclaim the fermentative production thing then with separatory membrane.
Background technology
Follow the material production method of the cultivation of microorganism or culturing cell, can roughly be divided into (1) batch culture method (Batch culture method) and fed batch cultivation method (Fed-Batch culture method), and (2) continuous culture method.
The batch culture method of above-mentioned (1) and the equipment of fed batch cultivation method are simple, cultivation finishes at short notice, therefore have the few advantage of infringement that living contaminants causes, and just use as the material production method of using microorganism or culturing cell all the time.But, these methods, because process in time, the fermentative production substrate concentration in the nutrient solution uprises, osmotic pressure rises or produces reasons such as fermentation that thing self causes is suppressed and causes productivity and yield to reduce.Therefore, adopt these cultural methods, be difficult to keep long-term and stably fermentative production object height productivity and high yield.
On the other hand, the continuous culture method of above-mentioned (2) therefore has and can keep high yield and the such feature of high productivity for a long time owing to avoided the interior fermentative production object height concentration of culture tank to accumulate.
For example, the fermentation for L-L-glutamic acid (with reference to patent documentation 1) and L-Methionin (with reference to non-patent literature 1) discloses continuous culture method.Yet, in these examples,, yet diluted microorganism or the culturing cell in the nutrient solution owing to taking out the nutrient solution that contains microorganism or culturing cell although carried out the continuous supply of raw materials such as nutrient substance to nutrient solution, therefore limited the raising of production efficiency.
Therefore, following method has been proposed, promptly in continuous culture method, with separatory membrane filtering microorganism or culturing cell, from see through liquid, reclaim the fermentative production thing, make the microorganism or the culturing cell that are filtered remain on or be back in the culture tank simultaneously, microorganism in the nutrient solution and cell are maintained high density.
For example, proposed, carried out the technology (with reference to patent documentation 2) of cultured continuously by utilizing the bactogen of separatory membrane.In this motion, has the groove that is used for culturing micro-organisms or culturing cell by use, from nutrient solution purpose fermentative production thing and microorganism, culturing cell are carried out the bactogen of the groove of membrane sepn with being used for, can be to produce various chemical than the production rate of culture method, fed batch cultivation Fa Genggao in batches.
Here, in the bactogen of use separatory membrane,, it is believed that therefore can make separatory membrane see through the liquid measure increase further improves production rate owing to make the nutrient solution flow velocity in the membrane sepn groove improve the obstruction that is not easy to cause film.But in patent documentation 2, because the liquor charging amount of can't independent control being sent by culture tank and to the flow of membrane sepn groove, the flow that therefore supplies to the nutrient solution of membrane sepn groove depends on the flow by the nutrient solution of culture tank liquor charging.Therefore, change, the liquor charging amount of being sent by culture tank is changed in order to make the nutrient solution flow velocity in the membrane sepn groove, the result, the liquid mixing state in the culture tank can change, and big the variation takes place in culture condition.In addition, process in time, film stops up, when reasons such as the concentration rising of microorganism or culturing cell cause membrane sepn groove internal pressure to rise, in order to make membrane sepn self optimization, the preferred flow that reduces the nutrient solution that supplies to the membrane sepn groove.But in case the flow of the nutrient solution that supplies to the membrane sepn groove is changed, then big variation will take place in the culture condition in the culture tank.Therefore, the flow of the nutrient solution that supplies to the membrane sepn groove is changed.In addition, if for the pressure in the controlling diaphragm separator tank the most suitably and reduce the cultivation liquid measure of sending by culture tank, the nutrient solution flow velocity that will take place in the liquid pushing tube reduces, and microorganism or culturing cell are deposited in the liquid pushing tube pipe arrangement, and production efficiency reduces such problem.On the other hand, if the hypertonia in the membrane sepn groove, with regard to having by the microorganism in the nutrient solution that outwards moves in the membrane sepn groove because pressure variation and destroyed such worry.
The prior art document
Patent documentation
Patent documentation 1: the spy opens flat 10-150996 communique
Patent documentation 2: the international brochure that discloses No. 07/097260
Non-patent literature
People such as non-patent literature 1:Toshihiko Hirao, Appl.Microbiol.Biotechnol., 32,269-273 (1989)
Summary of the invention
The problem that invention will solve
The present invention is in view of such situation, the manufacture method of chemical and the culture apparatus that can be suitable for this method are provided, this manufacture method can not influence the interior nutrient solution flow velocity of culture condition ground controlling diaphragm separator tank in the culture tank, but also can suppress the precipitation of microorganism or culturing cell, improve the production efficiency of chemical.
The means that are used to deal with problems
The inventor is with the raising of the production rate in the bactogen that utilizes separatory membrane and the stable purpose that turns to of fermentation culture, carried out positive research, found that, by each formation of following (1)～(14), the nutrient solution flow velocity in the controlling diaphragm separator tank on one side, keep the culture condition (residence time of nutrient solution etc.) in the culture tank on one side, can carry out effective manufacturing of chemical, thereby finish the present invention.
(1) makes a part by the nutrient solution of culture tank liquor charging, nutrient solution inflow side pressure according to the membrane sepn groove, walk around the manufacture method of the chemical of membrane sepn groove, this method is culturing micro-organisms or a culturing cell in culture tank, carry nutrient solution by culture tank to the membrane sepn groove, filter with separatory membrane again, from see through liquid, reclaim fermentative production thing, the nutrient solution that do not filter of filtered is refluxed in the mode that the nutrient solution with membrane sepn groove upstream side converges as chemical.
(2) manufacture method of the chemical described in aforementioned (1), its gauge pressure with the nutrient solution inflow side of membrane sepn groove are such below the 1MPa, and the flow of the nutrient solution of membrane sepn groove is walked around in control.
(3) manufacture method of aforementioned (1) or (2) described chemical, its make a part of not filtering nutrient solution with culture tank in the mode converged of nutrient solution reflux, make simultaneously the rest part that do not filter nutrient solution with and culture tank to the mode that the nutrient solution between the membrane sepn groove converges reflux.
(4) manufacture method of aforementioned (3) described chemical, its control independently of each other with and culture tank to the nutrient solution between the membrane sepn groove converge the flow that does not filter nutrient solution that mode refluxes and with culture tank in nutrient solution converge the flow that does not filter nutrient solution that mode refluxes.
(5) manufacture method of the chemical described in aforementioned (3) or (4), with and culture tank in nutrient solution converge the flow that does not filter nutrient solution that mode refluxes with respect to and culture tank to the nutrient solution between the membrane sepn groove converge the ratio of the flow that does not filter nutrient solution that mode refluxes below 1.
(6) manufacture method of each described chemical of aforementioned (1)～(5), the linear flow speed of the linear flow speed of the nutrient solution of being carried to the membrane sepn groove by culture tank, the linear flow speed of not filtering nutrient solution that refluxes in the mode of being converged to the nutrient solution of this membrane sepn groove upstream side by the membrane sepn groove and the nutrient solution of walking around the membrane sepn groove are all at 2.5cm/ more than second.
(7) manufacture method of each described chemical of aforementioned (1)～(6), regulate to flow into the liquid measure that sees through of the cultivation liquid measure of membrane sepn groove and/or self-separation film, so that the liquid measure that sees through of self-separation film is below 10.0% with respect to the rate of recovery of the cultivation liquid measure that flows into the membrane sepn groove.
(8) manufacture method of each described chemical of aforementioned (1)～(7), in the culture tank in nutrient solution volume and the membrane sepn groove ratio of nutrient solution volume be more than 4 below 100.
(9) possesses the bypass pipe of membrane sepn groove, the proofing unit of the nutrient solution inflow side pressure of membrane sepn groove, bactogen with the flow rate control device that is arranged on bypass pipe, this bactogen possesses the culture tank that is used for culturing micro-organisms or culturing cell, the membrane sepn groove that possesses the separatory membrane that is used for retrieving the fermentative production thing produced from the nutrient solution of culture tank, connect culture tank and membrane sepn groove and when nutrient solution is transported to the aforementioned films separator tank, make without filtering circulation tube that mode that nutrient solution converges with the nutrient solution with membrane sepn groove upstream side refluxes and the nutrient solution liquid feeding device that is arranged on circulation tube of not filtering of separatory membrane.
(10) bactogen described in aforementioned (9), described flow rate control device are to be turned round by result's interlock of proofing unit.
(11) bactogen described in aforementioned (9) or (10), it possesses the linear flow speed detector of circulation tube, and flow rate control device and/or aforementioned nutrient solution liquid feeding device are that the result according to this linear flow speed detector turns round.
(12) each described bactogen of aforementioned (9)～(11), described membrane sepn groove be installed in have the liquid feeding device different with aforementioned nutrient solution liquid feeding device and with culture tank independently in the circulation loop.
(13) each described bactogen of aforementioned (9)～(12), the position opening in the nutrient solution that circulation tube retains in impregnated in culture tank.
(14) each described bactogen of aforementioned (9)～(13), the long-pending ratio of culture tank volume and membrane sepn cell body is more than 4 below 100.
The invention effect
According to the present invention, can make by the part of the nutrient solution of culture tank liquor charging nutrient solution inflow side pressure, walk around the membrane sepn groove, that is to say according to the membrane sepn groove, the flow of the nutrient solution that can independently control the flow of the nutrient solution of supplying with to the membrane sepn groove and send by culture tank.Consequently, can not change the nutrient solution flow velocity in the culture condition ground appropriate change membrane sepn groove, and be not easy to cause the obstruction of film, can increase separatory membrane to see through liquid measure, thereby improve production rate.In addition, if, in time through the obstruction of film takes place, the concentration of microorganism or culturing cell rises, then the pressure in the membrane sepn groove rises, culture condition in the culture tank does not change substantially yet, not filtering the filtration in the circulation tube that nutrient solution refluxes of when nutrient solution is transported to the membrane sepn groove, refluxing without separatory membrane, microorganism can be kept on one side or culturing cell is difficult to sedimentary flow velocity, control on one side supplies to the flow of the nutrient solution of membrane sepn groove, and the pressure that in the membrane sepn groove, acts on, consequently can prevent the breakage of membrane sepn groove, prevent the destruction that the pressure change of microorganism in the nutrient solution or culturing cell causes.And, when in the membrane sepn groove, producing trouble,,, revise the trouble in the membrane sepn groove Yi Bian stop the supply of nutrient solution fully to the membrane sepn groove on one side can continue to cultivate, carry out the exchange or the conversion of membrane sepn groove.
In addition, among the present invention, get around the membrane sepn groove by making on one side according to the pressure of the nutrient solution inflow side of membrane sepn groove by the part of the nutrient solution of culture tank liquor charging, on one side the rate of recovery that sees through liquid in this membrane sepn groove is controlled at below 10%, the obstruction of film can be further prevented, cultured continuously can be carried out for a long time.
As mentioned above, according to the present invention, the production efficiency of the fermentative production thing (that is to say desired chemical) that cultured continuously produces can be improved simultaneously and to sugared yield, and, cultured continuously can also be carried out for a long time by the rate of recovery in the membrane sepn groove is controlled at below 10%.
Brief description of drawings
[Fig. 1] is the summary mode chart of an embodiment that is used for illustrating the present invention's bactogen.
[Fig. 2] is the summary mode chart of other embodiment that is used for illustrating the present invention's bactogen.
[Fig. 3] is the summary stretch-out view of an embodiment of the separating film element that is used for illustrating that the present invention uses.
[Fig. 4] is the summary oblique drawing of other embodiment of the separating film element that is used for illustrating that the present invention uses.
[Fig. 5] represents the yeast that uses in the reference example figure of the physical map of expression vector pTRS11.
Nutrient solution linear flow speed and the figure that is deposited in the thalline semi-invariant in this pipe in the circulation tube that obtains among [Fig. 6] embodiment 2.
[Fig. 7] is the summary mode chart of other embodiments that is used for illustrating the present invention's bactogen.
[Fig. 8] is the summary mode chart of other embodiments that is used for illustrating the present invention's bactogen.
[Fig. 9] is the summary mode chart of the scheme of the bactogen that is used for illustrating that comparative example uses.
[Figure 10] is the lactic acid concn that obtains among the expression embodiment 1, the figure of yeast turbidity.
[Figure 11] is the lactic acid concn that obtains in the expression comparative example 1, the figure of yeast turbidity.
[Figure 12] is the figure of the pressure of the nutrient solution inflow side of the membrane sepn groove of acquisition in the expression comparative example 1.
[Figure 13] is the summary mode chart of the scheme of the bactogen that is used for illustrating that comparative example uses.
[Figure 14] is the summary mode chart of other embodiment that is used for illustrating the present invention's bactogen.
[Figure 15] is the summary mode chart of the scheme of the bactogen that is used for illustrating that comparative example uses.
[Figure 16] is the summary mode chart of other embodiment that is used for illustrating the present invention's bactogen.
[Figure 17] is the figure of the passing of the intermembranous differential pressure of acquisition among the expression embodiment 6～9.
[Figure 18] is the cadaverine concentration that obtains among the expression embodiment 10, the figure of excellent bacillus turbidity.
[Figure 19] is the cadaverine concentration that obtains in the expression comparative example 5, the figure of excellent bacillus turbidity.
[Figure 20] is the figure of the pressure of the nutrient solution inflow side of the membrane sepn groove of acquisition in the expression comparative example 5.
[Figure 21] is the L-lysine concentration that obtains among the expression embodiment 11, the figure of excellent bacillus turbidity.
[Figure 22] is the L-lysine concentration that obtains in the expression comparative example 6, the figure of excellent bacillus turbidity.
[Figure 23] is the figure of the pressure of the nutrient solution inflow side of the membrane sepn groove of acquisition in the expression comparative example 6.
The best mode that is used to carry out an invention
Method of the present invention is the manufacture method of chemical, it is culturing micro-organisms or culturing cell in culture tank, nutrient solution is transported to the membrane sepn groove continuously by culture tank, filter with separatory membrane, reclaim the fermentative production thing as chemical from see through liquid, the nutrient solution nutrient solution that makes it with the upstream side of membrane sepn groove that refluxes that do not filter of filtered is converged, this moment is by the part of the nutrient solution of culture tank liquor charging, nutrient solution inflow side pressure according to the membrane sepn groove gets around the membrane sepn groove.
Such the present invention carries out at culture apparatus for example shown in Figure 1.Fig. 1 is the summary mode chart of this culture apparatus in one embodiment of the invention.
Culture apparatus shown in Fig. 1 by the culture tank 1 of the cultivation of carrying out microorganism or culturing cell and the membrane sepn groove 2 that possesses the separatory membrane 3 that is used to filter nutrient solution constitute.Membrane sepn groove 2 is arranged on the outside of cultivation reaction bath, is connected with culture tank 1 with liquid pushing tube 15 (circulation tube) by liquid pushing tube 17.
Culture tank 1 has can the cultured continuously microorganism or the function of culturing cell, as long as can connect circulation tube, it can be an arbitrary structures, can use pot type fermentation container that in the past always is used for culturing micro-organisms or culturing cell etc.
Culture tank 1 constitutes as follows, and it is connected with substratum supply pump 6, and is equipped with stirrer 7, by substratum supply pump 6 substratum is put in the culture tank 1, as required, can use the nutrient solution in the stirrer 7 stir culture grooves 1.And, also connect gas supply device 8, as required, supply with the gas that needs by this gas supply device 8.At this moment, also preference as, connecting pipings between the head space (headspace) of culture tank 1 and gas supply device 8, press the mobile supply gas of order of head space, pipe arrangement, gas supply device 8, thereby can reclaim and the recirculation gas supplied, supply with by gas supply device 8 again.
In addition, pH sensor control 9 and pH regulator fluid supply pump 10 are set as required in culture tank 1, so that can regulate the pH of nutrient solution.Certainly, the pH in order to provide bronsted lowry acids and bases bronsted lowry two sides to control nutrient solution when cultivating preferably has a plurality of pH regulator fluid supply pumps.And, as required, thereby thermoswitch 11 also is set so that can regulate the production that culture-liquid temp carries out the chemical of high productivity.And, adjusting as the physical and chemical condition of the nutrient solution that is undertaken by instrument detecting and control device, illustration the adjusting of pH and temperature, but as required, can also carry out the control of dissolved oxygen and ORP, measure microorganism concn in the nutrient solution but also can be used in analytical equipment such as linearize transmitter again, control is with its physical and chemical condition as index.And the measured value of the physicochemical environment of the nutrient solution that obtains with above-mentioned instrument detecting and control device can suitably be regulated substratum input amount and speed as index.
In membrane sepn groove 2 inside separatory membrane 3 can be set, membrane sepn groove 2 is same with culture tank 1, as long as can connect circulation tube, its shape etc. is not limit.If separatory membrane 3 can only not filter microorganism or culturing cell from the nutrient solution of microorganism or culturing cell, no matter be inorganic or the separatory membrane of organic materials can, but preferably have the proterties that is suitable for processed liquid and the separation performance and the diactinic porous membrane described later of purposes, and the film of preferred ability disinfecting action (for example 120 ℃ 30 minutes).And pump 4 is connected with separatory membrane 3, so that can produce intermembranous differential pressure in the stoste side of this separatory membrane with through between the side.
And preferred, the nutrient solution volumetric ratio that membrane sepn groove 2 and culture tank 1 have nutrient solution in the culture tank and a nutrient solution in the membrane sepn groove reaches more than 4 below 100.That is to say, preferably, be designed to that in membrane sepn groove 2 and culture tank 1 each groove, the general nutrient solution that stores their volumetrical about 80% that adds, the long-pending ratio of culture tank volume and membrane sepn cell body reach more than 4 below 100.By this structure, can in the densification of implement device, prolong the culture tank residence time of nutrient solution, realize suitable culture condition, and can reduce power charge, improve the production rate of chemical, and realize being easy to the device running management.
In circulation tube (liquid pushing tube 17 and liquid pushing tube 15), setting is by the nutrient solution inflow side of membrane sepn groove 2, walk around the membrane sepn groove, the bypass pipe 26 that is connected with the nutrient solution outflow side of membrane sepn groove, this structure makes it possible to not supply to membrane sepn groove 2 by the part of the nutrient solution of culture tank 1 conveying, make it to walk around this membrane sepn groove 2, and converge with the not filtration nutrient solution of liquid pushing tube 15.In addition, in the present embodiment, one end of bypass pipe 26 is connected with liquid pushing tube 17, the other end is connected with liquid pushing tube 15, but bypass pipe 26 also can be from the nutrient solution inflow side of membrane sepn groove 2, walk around membrane sepn groove 2, with culture tank 1 or with the nutrient solution inflow side of culture tank 1 and membrane sepn groove 2 between be connected.That is to say that for this a part of nutrient solution of walking around membrane sepn layer 2 directly is back to culture tank 1, also an end (upstream side) of bypass pipe 26 can be connected with liquid pushing tube 17, the other end (downstream side) is connected with culture tank 1.In addition, the two ends of bypass pipe 26 can also be connected with liquid pushing tube 17, so that the nutrient solution of walking around in the liquid pushing tube 17 that this part nutrient solution of membrane sepn layer 2 is direct and culture tank 1 is supplied with converges.
In the bypass pipe 26 of membrane sepn groove 2, has flow rate control device 25.By this flow rate control device, can control the nutrient solution flow of supplying with to membrane sepn groove 2.This flow rate control device can be a valve, also can be pump, but considers preferred valve from the cost aspect.When selector valve during, can reduce the cultivation liquid measure that flows to membrane sepn groove 2 by driving valve as aforementioned flow rate control device.In addition, on the contrary,, can make all nutrient solutions that flow through liquid pushing tube 17 flow to membrane sepn groove 2 by closing valve.Although the structure of valve is not limit, because when steam sterilizing, nutrient solution etc. are structurally residual few, therefore preferred diaphragm valve (diaphragm valve), the butterfly valve of using.In addition, when selecting pump as flow rate control device 25, by by nutrient solution with flow to the equidirectional such liquor charging that flows of the nutrient solution of membrane sepn groove 2, increase the pumping liquid measure, can reduce the cultivation liquid measure that flows to membrane sepn groove 2, on the contrary, by stopping pumped liquid, can make the nutrient solution that flows through liquid pushing tube 17 all flow to membrane sepn groove 2.
The flow of the nutrient solution of supplying with to membrane sepn groove 2 is controlled according to the nutrient solution inflow side pressure of membrane sepn groove basically.For this reason, pressure warning unit 29 is set in device shown in Figure 1.Nutrient solution inflow side pressure with pressure warning unit 29 mensuration membrane sepn grooves when measured value is higher than desirable value, operationalizes flow rate control device 25, makes the part of the nutrient solution of being carried by culture tank 1 walk around 2 circulations of membrane sepn groove.
In addition, in circulation tube, be provided with the pump 5 that carries out by the flow control of the nutrient solution of culture tank liquor charging.Pump can be arranged in the liquid pushing tube 17 or in the liquid pushing tube 15 (returning culture tank), also can be arranged in both.The mode of pump, shape and connect liquid portion material etc. and do not limit, but the material of steam sterilizing that preferably can anti-circulation tube.
At this paper, represent the nutrient solution linear velocity in the circulation tube among Fig. 6 and had relation between the precipitation capacity of yeast strains of lactic acid-producing ability, show thus, in the circulation tube (liquid pushing tube 17 and liquid pushing tube 15) if the nutrient solution linear velocity at 2.5cm/ more than second, thalline just can not be deposited in the pipe arrangement, and nutrient solution is circulated.Therefore, detect, flow rate control device 25 and pump 5 are operationalized, so that this linear flow speed reaches 2.5cm/ more than second by nutrient solution in the liquid pushing tube 17 of culture tank conveying and/or the linear flow speed of not filtering nutrient solution in the liquid pushing tube 15.And, based on identical reason, preferably manage to make the linear flow speed of nutrient solution in the bypass pipe 26 to reach 2.5cm/ more than second.
And, as mentioned above, nutrient solution in the aforementioned part nutrient solution of walking around the membrane sepn groove and culture tank or when converging by the nutrient solution that culture tank is carried to the membrane sepn groove, detection is by the linear flow speed of the nutrient solution of culture tank liquor charging, flow rate control device 25 and pump 5 are operationalized, so that this linear flow speed reaches 2.5cm/ more than second.In addition, as described later, liquid pushing tube 15 do not filter nutrient solution both by with culture tank in the mode circulation that converges of nutrient solution, during again by the mode circulation that directly converges with the part of the nutrient solution of liquid pushing tube 17, preferably manage to make the linear flow speed of the nutrient solution in arbitrary these pipes all to reach 2.5cm/ more than second.That is to say, among the present invention, preferably manage to make from culture tank be transported to the linear flow speed of the nutrient solution of membrane sepn groove, the linear flow speed of not filtering nutrient solution that refluxes in the mode of converging to the nutrient solution of the upstream side of this membrane sepn groove from the membrane sepn groove and the linear flow speed of walking around the nutrient solution of membrane sepn groove all reaches 2.5cm/ more than second.
And, in device shown in Figure 1,, water level sensor 12 is set in culture tank 1 in order to regulate the cultivation liquid measure that filters in the separatory membrane 3 in flow and the culture tank.By the cultivation liquid measure that water level sensor 12 detects in the culture tank, control substratum supply pump 6.In addition, in order to regulate the filtration flow, can control through liquid measure.The method that control sees through liquid measure does not have restriction, changes the waterhead pressure control device that filters flow but for example can be provided with by control water level differential pressure, changes the filtration flow thereby can also pump be operationalized by power supply power.In addition, it is desirable to, be provided for steam suppling tube in the culture apparatus that in the manufacture method of chemical of the present invention, uses to culture tank 1, membrane sepn groove 2, liquid pushing tube 15, liquid pushing tube 17 sterilizations.
In addition, as the various pumps that use among the present invention, can enumerate for example various kinds such as spiral pump, pipe pump, surge pump, but preferably set according to the output of pump, can the computation cycles liquid measure or the pump that sees through liquid measure of self-separation film, specifically can enumerate surge pump or pipe pump.
In the manufacture method of the chemical of the culture apparatus that adopts above structure, cultivate and undertaken by for example following mode.That is to say, cultured continuously microorganism or culturing cell in culture tank 1, nutrient solution is from culture tank 1, by the pump in the circulation tube 5, through liquid pushing tube 17, supply with to membrane sepn groove 2, produce pressure difference by pump 4 grades in the stoste side of separatory membrane 3 and through between the liquid side, filter this nutrient solution, recovery contains the liquid that sees through as (chemical) such as lactic acid of the fermentative production thing of microorganism or culturing cell.On the other hand, do not filter nutrient solution, be back in the culture tank 1 through liquid pushing tube 15.At this moment, the flow of pump 5 is not deposited in speed in liquid pushing tube 17 and the liquid pushing tube 15 (for example aforesaid linear flow speed at 2.5cm/ more than second) liquor charging with microorganism or culturing cell.
Yet if proceed cultivation, membrane sepn continuously, the viscosity of nutrient solution rises, and separatory membrane stops up and interior microorganism of membrane sepn groove or culturing cell accumulation, and reasons such as stream obturation cause the interior pressure of membrane sepn groove to rise.Membrane sepn groove internal pressure is in case rise, and increased the damaged of membrane sepn groove and to the burden of microorganism or culturing cell.Therefore, the pressure in the membrane sepn groove is preferably below 1MPa.On the other hand, if rise in order to suppress membrane sepn groove internal pressure, thereby the nutrient solution liquor charging amount that is provided by culture tank 1 by pump 5 is provided, big variation will take place in the culture condition in the culture tank, and microorganism or culturing cell be deposited in the circulation tube, and production efficiency reduces.
Therefore, among the present invention, make the part of the nutrient solution of carrying by culture tank 1,, walk around membrane sepn groove 2, reflux according to the nutrient solution inflow side pressure of membrane sepn groove 2.Preferably, the pressure of nutrient solution inflow side of pressing the membrane sepn groove is controlled the flow of the nutrient solution of walking around membrane sepn groove 2 below 1MPa.In addition, so-called here this pressure is gauge pressure, so-called pressure among the present invention, and short of specified otherwise is just represented gauge pressure.Pressure variation in this membrane sepn groove can be measured by the pressure warning unit 29 that is arranged on the nutrient solution supply side, and according to this measurement result, the flow of the nutrient solution of membrane sepn groove is walked around in control, thereby the pressure in the controlling diaphragm separator tank rises.Consequently, can prevent the precipitation of microorganism in the circulation tube or culturing cell, stably cultivate, and can reduce the damaged of membrane sepn groove and the burden of microorganism, culturing cell is increased, therefore can obtain high yield and high productivity.That is to say that compare with batch fermentation in the past, the production rate of fermentative production thing is higher, can carry out the fabulous fermentative production of efficient, simultaneously can this fermentative production thing of efficient recovery.And the production rate in the cultured continuously is calculated with following formula (1).
[several 1]
Figure BDA0000052877920000121
In addition, the fermentative production speed in the batch culture is obtained in the following manner: the production thing amount (g) during with all raw material carbon sources of consumption consumes needed time (h) and this time point divided by carbon source cultivation liquid measure (L).
Device shown in Figure 1 preferably changes by for example following mode.That is to say that for example, as shown in Figure 2, preferably press the measurement result interlock of flow rate control device 25 and pressure warning unit 29, the mode that operates constitutes.In addition, preferably in liquid pushing tube 17, in the downstream side of the tie point of bypass pipe 26 and the position of membrane sepn layer 2 upstream side, membrane sepn groove open and close valve 27 is set, in liquid pushing tube 15, on the position in the upstream side of the tie point of bypass pipe 26 and membrane sepn layer 2 downstream side, membrane sepn groove open and close valve 28 is set.When membrane sepn groove open and close valve 27 and/or membrane sepn groove open and close valve 28 are set, manage to make as required, to flow to bypass pipe 26 by all nutrient solutions that flow through liquid pushing tube 17.So, in the obstruction of separatory membrane and membrane sepn groove the accumulation of microorganism or culturing cell cause producing under the situation such as stream obturation in the membrane sepn groove uncomfortable in, can stop nutrient solution fully, can revise or the interior uncomfortable place of exchange membrane separator tank to the supply of membrane sepn groove.
And, also preferred, as shown in Figure 7, liquid pushing tube 15 do not filter the mode circulation that the nutrient solution in nutrient solution and the culture tank converges in, by the mode circulation that directly converges with the part of the nutrient solution of liquid pushing tube 17.At this moment, control by with culture tank in the flow velocity that does not filter nutrient solution, flow of the mode circulation that converges of nutrient solution and the downstream of culture tank side that is arranged on the tapping point 14B of liquid pushing tube 15 by the pump 5 of the flow velocity of the nutrient solution of culture tank liquor charging, flow, in addition, also pump 16 is set in the downstream side of the point 14A of liquid pushing tube 17.So, upstream side at the tapping point 14B of the downstream side of the point 14A of membrane sepn groove 2, liquid pushing tube 17 and liquid pushing tube 15, formation and culture tank 1 be recycle circuit independently, can realize respectively being controlled at flow velocity in the circulation loop that the upstream side of the tapping point 14B of the downstream side of point 14A of membrane sepn groove 2, liquid pushing tube 17 and liquid pushing tube 15 forms, flow independently of each other and at flow velocity, the flow of the upstream side of the point 14A of downstream side, culture tank 1 and the liquid pushing tube 17 of the tapping point 14B of liquid pushing tube 15 by pump 16,5.Therefore, even regulating pump 16, increase the nutrient solution flow velocity in this circulation loop, that is to say, make under the situation of linear velocity (linear flow speed) of nutrient solution on the surface of flowing through the separatory membrane 3 in the membrane sepn groove, also can make the flow velocity in downstream side of the tapping point 14B of liquid pushing tube 15 keep certain by pump 5, the speed that nutrient solution is returned in the culture tank 1 keeps certain.That is to say, owing to the nutrient solution flow velocity in the membrane sepn groove is improved with can keeping the interior culture condition of culture tank, therefore can be with microorganism or the non-setting speed liquor charging of culturing cell, and can keep being suitable for culture condition in the culture tank, can stably cultivate, can keep high yield and high productivity.
In addition, in a single day the culture tank 1 interior speed of not filtering nutrient solution that turns back to nutrient solution accelerate, and will become the essential factor of liquid rheology unrest, influences oxygen and move capacity coefficient kLa, the speed that nutrient solution is turned back in the culture tank 1 keeps certain, thereby makes the fermentation efficiency stabilization.
And, because the flow velocity on separatory membrane 3 surfaces in the membrane sepn groove is flow through in increase of the present invention, what have increased access to sees through liquid, it is the yield of fermentative production thing, and maintenance culture efficiency, improve production rate, therefore by with culture tank in the flow that does not filter nutrient solution that refluxes of the mode converged of nutrient solution, flow velocity (just, the flow in the downstream side of the tapping point 14B of liquid pushing tube 15, flow velocity) α preferably than by and the flow that does not filter nutrient solution that refluxes of the mode converged to the nutrient solution between the membrane sepn groove of culture tank, flow velocity (the flow in the downstream side of the tapping point 14A of liquid pushing tube 17 just, flow velocity) β is little, and their ratio α/β is preferably below 1.
And as shown in figure 14, the liquid pushing tube 15 of the mode circulation that converges by the nutrient solution that does not filter in nutrient solution and the culture tank preferably impregnated in the position opening of the nutrient solution that is stored in culture tank 1.Because an end of liquid pushing tube 15 is at this position opening, the oxygen in the culture tank 1 moves capacity coefficient kLa and is difficult to set point change by expection, the decrement from the set(ting)value of this coefficient can be suppressed at this set(ting)value 30% with interior scope.
In addition, as shown in Figure 8, preferred a plurality of membrane sepn grooves 2 are connected in parallel.In this way, when generation is inappropriate in a membrane sepn groove, can changes the membrane sepn groove and re-use, can not stop filtration, proceed to cultivate with the membrane sepn groove.In addition, when being connected in parallel a plurality of membrane sepn groove,, can carry out the sterilization in the membrane sepn groove respectively by steam suppling tube being connected on each membrane sepn groove.
Among the present invention, in order to improve the yield of fermentative production thing, preferably prevent the obstruction of separatory membrane, stably the long term maintenance cultured continuously.Therefore, preferably be controlled at below 10.0% with respect to the rate of recovery of the ratio of the flow of the nutrient solution that is transported to the membrane sepn groove (below, only be called the rate of recovery sometimes) as the flow that sees through liquid that obtains by separatory membrane 3.
The so-called rate of recovery is the ratio of liquid measure with respect to the cultivation liquid measure (circulating fluid volume) that is transported to the membrane sepn groove that see through of time per unit separatory membrane 3, calculates by following (formula 2).But, when connecting a plurality of membrane separation unit, according to calculating in each membrane separation unit through liquid measure and circulating fluid volume.And seeing through liquid measure can replace with the filtration flow of the separatory membrane area that uses in the membrane separation unit and the control of can turning round, and (formula 2) can be converted into following (formula 3).
[several 2]
Figure BDA0000052877920000151
[several 3]
Figure BDA0000052877920000161
In order to control the rate of recovery, can regulate the cultivation liquid measure that flows into the membrane sepn groove and/or see through liquid measure by what separatory membrane obtained.That is to say, preferably to being selected from circulating fluid volume, filtering flow and seeing through controlling more than a kind in the liquid measure.For the Control Circulation liquid measure, preferably regulate the aforementioned output that is positioned at the pump 5,16 of membrane sepn groove upstream side.And as controlling the method that filters flow or see through liquid measure, the output of preferred pump 4 is regulated.
Therefore, in device shown in Figure 1, for example, seeing through in the liquid take-off pipe of liquid pushing tube 17 and separatory membrane 3 under meter is set, regular monitoring circulating fluid volume and see through liquid measure, with (formula 2) calculate recovery rate, preferably reach such below 10.0% by the rate of recovery, in control pump 4,5 outputs, turn round.
And, as controlling the method that filters flow or see through liquid measure, except the output of pump 4 is regulated, can also enumerate the adjusting of water level differential pressure, the attraction of passing through generations such as liquid or gas or the pressurization in the membrane sepn groove etc.
By these methods, just for example can realize that to keep certain circulating fluid volume, only the mode of control filtration flow turns round simultaneously.In addition, can also realize that the mode of Control Circulation liquid measure turns round simultaneously to keep certain filtration flow.
The rate of recovery more preferably is controlled at below 5.0%.On the other hand, consider that from improving this viewpoint of energy efficiency the rate of recovery is high more good more.Therefore, the lower limit of the rate of recovery is preferably at least more than 0.01%.
Below, describe filtering flow.The so-called flow that filters can calculate with following (formula 4).
[several 4]
Figure BDA0000052877920000171
The membrane area that uses in the device can be set arbitrarily, is clearly therefore.See through the volume (m of liquid measure 3/ day) preferably measure the volume that saw through liquid measure in 1 day, but can also come 1 day the volume that sees through liquid measure of budgetary estimate by the volume of measuring about 1 hour that sees through liquid measure.Among the present invention, preferably filter flow below 0.500m/ days, more preferably 0.050m/ days above below 0.400m/ days.Filter flow in case surpass 0.500m/ days, be difficult to stably keep cultured continuously sometimes by the control of the rate of recovery.In addition, if filter flow less than 0.050m/ days, this just means that the separatory membrane area is excessive, considers from the viewpoint of economy, is difficult to industrialization.
Below, an example that carries out the manufacturing of chemical with the device shown in Fig. 1 specifies as follows.
At first, microorganism and cultivate raw material (substratum) and be stored in culture tank 1, by suitable interpolation neutralizing agent, in the scope of pH4～8, simultaneous temperature maintains 20～50 ℃ scope with the inner sustain of this culture tank 1.Thus, carry out the cultivation of microorganism, produce desirable fermentative production things (chemical) such as alcohol, organic acid, amino acid and nucleic acid this moment.During this,, obtain desirable fermentative production thing, by substratum supply pump 6, to culture tank 1 successive or the substratum that contains nutrient substance that uses in supply with cultivating off and in order to cultivate continuously.
In addition, when cultivating, make the linear flow speed in the circulation tube reach 2.5cm/ more than second, make the nutrient solution circulation continuously between culture tank 1 and membrane sepn groove 2 in the culture tank 1 with pump 5.In membrane sepn groove 2, by separatory membrane this nutrient solution is filtered and is separated into and contain not filtering nutrient solution and containing the liquid that sees through of fermentative production thing of microorganism.Consequently, take out the liquid that sees through that contains the fermentative production thing from apparatus system, reconcentration, distillation, partial crystallization should see through liquid, can obtain the fermentative production thing that purity improves.On the other hand, rest in the culture tank 1 with the isolating not filtration nutrient solution that contains microorganism or culturing cell after filtration, so the microorganism of culture tank can maintain high density, can carry out the cultivation of the high productivity of chemical.
And the calculating of the linear flow speed in the circulation tube can be calculated by (flow of each unit time)/(sectional area of pipe arrangement).Perhaps can in pipe arrangement, connect Corioli formula digitizing flow sensor or electrode non-contact type 2 line formula magnetic flow meters etc., measure.Survey the output of these digitizing under meters, carry out the control of pump 5 or flow rate control device 25 etc.
In the high scope of the constant rate that microorganism in the nutrient solution in the culture tank 1 or culturing cell concentration, the propagation of preferred microorganism or culturing cell become incompatible and dead, maintain high state, this is in order to obtain high efficiency productivity.As an example,, can obtain good production efficiency by concentration is maintained dry weight more than 5g/L.
In order to keep this suitable concentration, also preferably as required, in culture tank, take out microorganism or culturing cell.If the situation of separatory membrane obturation will appear taking place easily in microorganism or culturing cell excessive concentration in the culture tank sometimes.Maintain suitable concentration by taking-up, can avoid the obturation of separatory membrane.And, because the microorganism in the culture tank or the concentration of culturing cell cause the production performance of chemical to change sometimes, therefore by with production performance as index, take out microorganism or culturing cell, production performance can also be maintained within a certain range.
Cultivating the supply of raw material and the taking-up (nutrient solution is to the liquor charging of membrane sepn groove) of nutrient solution can begin from suitable period to carry out.That is to say that cultivating the supply of raw material and the beginning of nutrient solution taking-up may not need at the same time period.And cultivating the supply of raw material and the taking-up of nutrient solution can be successive, also can be intermittently.
And, also preferred by water level sensor 12 as required, suitably regulate the cultivation liquid measure in the culture tank.In the adjusting of the cultivation liquid measure in the culture tank, can also not the cultivation liquid level of measuring in the culture tank, regulate but measure nutrient solution weight.
Among the present invention, get final product so long as can generate chemical in microorganism or culturing cell propagation, the number of culture apparatus is not limit.The cultured continuously operation is considered from cultivating management, preferably carries out in single culture tank usually, but owing to reasons such as the culture tank capacity are little, also can use a plurality of culture tanks.At this moment, promptly use the in parallel or a plurality of fermenters that are connected in series of pipe arrangement, carry out cultured continuously, also obtained the high productivity of fermentative production thing.
And, among the present invention, so-called nutrient solution, be meant and cultivate the liquid that can access the result of microorganism or culturing cell propagation in the raw material, the so-called raw material of cultivating, be meant to promote growing of institute's cultured microorganism or culturing cell the nutrient substance of the chemical of the production of production purpose well thing etc. etc.For the composition of cultivating raw material, in order to improve the productivity of purpose chemical, the cultivation raw material composition in the time of also can beginning cultivation carries out appropriate change.
Microorganism of using among the present invention and culturing cell can be enumerated, for example bacterium, filamentous fungus, actinomycetes, zooblast, insect cells etc. such as the yeast such as bread yeast that can often use in industry, intestinal bacteria, rod-like stem bacterial type bacterium.Especially preferred, because isolated nuclei internal pressure differences and the eukaryotes such as yeast that cause cytoclasis easily, this is yeast most preferably wherein.The microorganism or the culturing cell that use can be isolating from physical environment, and, can also be because sudden change or gene recombination cause the change of part character.In these microorganisms or the culturing cell, preferably select the throughput of application target chemical high.And among the present invention, the cultivation with microorganism sometimes is called " fermentation " or " fermentation culture ".
As cultivating raw material, so long as promote growing of institute's cultured microorganism or culturing cell, the raw material of the chemical of production purpose well gets final product.As the object lesson of cultivating raw material, preferably use carbon source, nitrogenous source, inorganic salts and suitably contain the common liquid nutrient medium of organic micro-nutrientss such as amino acid, VITAMIN as required.
As carbon source, can also use carbohydrates such as glucose, sucrose, fructose, semi-lactosi, lactose, contain the alcohols such as organic acid, ethanol such as starch saccharificating liquid, sweet potato molasses, beet sirup, senior molasses (High Test Molasses) and acetic acid of these carbohydrates and glycerine etc.
As nitrogenous source, for example oil meal class, soybean add peptide class, various fermentation thalline such as water decomposition liquid, casein resolvent, other amino acid, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptone and add water decomposition thing etc. can to use ammonia, ammoniacal liquor, ammonium salt class, urea, Nitrates, other auxiliary organic nitrogen source that uses.
As inorganic salts, can suitably add phosphoric acid salt, magnesium salts, calcium salt, molysite and manganese salt etc.When needing microorganism growth to grow required specific nutrition element, can add nutrition as goods, perhaps contain this nutraceutical natural goods.And, can also use defoamer as required.
Among the present invention, the concentration of saccharide in the preferred nutrient solution remains on below the 5g/l.Why preferred this concentration of saccharide remain on below the 5g/L, is in order to make the loss of the carbohydrate that the taking-up of nutrient solution causes minimize.
And, the cultivation of microorganism or culturing cell, usually mostly in pH4～8, the scope of 20～50 ℃ of temperature carries out.The pH enough mineral acids of energy or organic acid, alkaline matter and urea, lime carbonate and the ammonia etc. of nutrient solution are adjusted to the predetermined value in the above-mentioned scope.And, improve the feed speed of oxygen if desired, can adopt and in air, add oxygen, oxygen concn is remained on more than 21%, or, improve stirring velocity pressurizeing in the cultivation reaction bath, improve means such as air flow.
Among the present invention, can carry out batch culture or fed batch cultivation, after the concentration of microorganism or culturing cell improves, begin cultured continuously, also can inoculate the thalline of high density, when cultivating beginning, carry out cultured continuously at the cultivation initial stage.
As the chemical (fermentative production thing) that the present invention can make,, without limits, can suitably select according to cultured microorganism or culturing cell so long as the material that aforementioned micro organism or culturing cell are produced in nutrient solution gets final product.As object lesson, can enumerate mass producible material in the fermentation industries such as alcohol, organic acid, amino acid, nucleic acid.As alcohol, can enumerate ethanol, 1, ammediol, 1,4-butyleneglycol, glycerine etc., as organic acid, can enumerate acetic acid, lactic acid, pyruvic acid, succsinic acid, oxysuccinic acid, methylene-succinic acid, citric acid, if nucleic acid can be enumerated diamine compounds such as Nucleotide such as nucleosides such as Trophicardyl, guanosine, hypoxanthylic acid, guanylic acid and cadaverine.And the present invention can also be applicable to materials such as producing enzyme, microbiotic, recombinant protein.
Below, to being used to obtain the microorganism or the culturing cell of desirable chemical, the limit is enumerated concrete chemical limit and is described.
Microorganism or culturing cell as using when the production lactic acid of the present invention are not particularly limited, but preferably can use milk-acid bacteria.Here alleged milk-acid bacteria can be defined as, and with respect to the glucose that consumes, produces the prokaryotic micro-organisms to the lactic acid of sugared yield more than 50%.As preferred milk-acid bacteria, can enumerate, for example, belong to lactobacillus (Lactobacillus), Pediococcus (Pediococcus), Tetracoccus (Tetragenococcus), carnivorous Bacillaceae (Carnobacterium), roaming Coccus (Vagococcus), leuconos toc (Leuconostoc), wine Coccus (Oenococcus), unusual Pseudomonas (Atopobium), streptococcus (Streptococcus), enterococcus spp (Enterococcus), lactococcus (Lactococcus), or the milk-acid bacteria of bacillus (Bacillus).In these milk-acid bacterias, select the milk-acid bacteria high of lactic acid, can be preferred for the production of lactic acid sugared yield.
And then, among the lactic acid, can also select the milk-acid bacteria high of the L-lactic acid in the lactic acid to sugared yield.So-called L-lactic acid is optical isomer a kind of of lactic acid, can clearly distinguish with its enantiomorph D-lactic acid.The milk-acid bacteria high as L-lactic acid to sugared yield, can enumerate for example fruit juice Bacterium lacticum (Lactobacillus yamanashiensis), Lactobacillus animalis (Lactobacillus animalis), Lactobacillus agilis (Lactobacillus agilis), birds Bacterium lacticum (Lactobacillus aviaries), lactobacterium casei (Lactobacillus casei), lactobacillus delbruckii (Lactobacillus delbruekii), lactobacillus paracasei (Lactobacillus paracasei), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus ruminis (Lactobacillus ruminis), lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus sharpeae (Lactobacillus sharpeae), dextrin sheet coccus (Pediococcus dextrinicus) or Lactococcus lactis (Lactococcus lactis) etc., can select them, be used for the production of L-lactic acid.
On the other hand, as microorganism that can be used for the D-lactic acid-producing or culturing cell, can enumerate lactobacillus delbruckii (Lactobacillus delbruekii), plant lactobacillus (Lactobacillus plantarum), pediococcus acidilactici (Pediococcus acidilactici), left lactic acid lactobacillus (Sporolactobacillus laevolacticus) or synanthrin lactobacillus (Sporolactobacillus inulinus) etc.
In addition, the present invention can adopt the microorganism or the culturing cell that have given artificially or strengthened the lactic acid-producing ability when making L-lactic acid or D-lactic acid.The method that gives artificially or strengthen the lactic acid-producing ability can be the known in the past method by the medicament variation, but preferably uses recombinant microorganism.As recombinant microorganism, preferably by import the L-lactate dehydrogenase gene (below, be also referred to as L-LDH) or D-lactate dehydrogenase gene (below, be also referred to as D-LDH), give or strengthened the L-lactic acid of microorganism or culturing cell or the recombinant microorganism of D-lactic acid-producing ability.
As the host of aforementioned recombinant microorganism, preferably as the intestinal bacteria of prokaryotic cell prokaryocyte, milk-acid bacteria with as eukaryotic yeast etc., special preferred yeast.The yeast that preferably belongs to yeast belong (Saccharomyces) in the yeast, more preferably yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
As L-LDH or D-LDH, nicotinamide adenine dinucleotide reduced (NADH) and pyruvic acid are converted into NAD (NAD so long as coding has +) and active proteinic L-serum lactic dehydrogenase or the D-serum lactic dehydrogenase of L-lactic acid or D-LDH, then without limits.This wherein, as L-LDH, preferably can end user (Homo sapiens) L-LDH in source or frog source.Among the L-LDH in frog source, the preferred L-LDH that uses the frog source that belongs to Pipidae (Pipidae) belongs in the frog of Pipidae, can preferably use the L-LDH in Africa xenopus (Xenopus laevis) source.In addition, as D-LDH, preferably the gene in plant lactobacillus (Lactobacillus Plantarum) and pediococcus acidilactici (Pediococcus acidilactici) or left lactic acid bacillus (Bacillus laevolacticus) source is more preferably the gene that left lactic acid bacillus is originated.
Comprise polymorphism among spendable L-LDH of the present invention or the D-LDH, bring out the gene of the mutant that sudden change etc. causes owing to heredity.The polymorphism of so-called heredity is that the base sequence part of the gene that causes owing to the spontaneous mutation on the gene changes.And what is called is brought out sudden change, is meant that artificial the importing suddenlyd change in gene.Bringing out suddenlys change the method for for example using the special sudden change importing in position to use test kit (Mutan-K (takara bio society system)), the method that test kit (BD Diversify PCR Random Mutagenesis (CLONTECH corporate system)) is used in the importing of employing random mutation etc.And L-LDH that uses among the present invention or D-LDH also can exist disappearance or insertion as long as coding has the protein of L-serum lactic dehydrogenase or D-lactate dehydrogenase activity in the part of base sequence.
And when making L-lactic acid, the separation and the purifying that see through L-lactic acid contained the liquid that obtain from above-mentioned separatory membrane 3 can knownly in the past concentrate by combination, methods such as distillation and partial crystallization carry out.For example, can enumerate by with the seeing through below the pH regulator to 1 of liquid of separatory membrane 3, the method for extracting with diethyl ether or ethyl acetate etc. then; Be adsorbed in ion exchange resin, clean the method for back wash-out; In the presence of acid catalyst, make it and the alcohol reaction, as ester distillatory method; And crystallization is the method for calcium salt or lithium salts etc.Preferably, can enumerate the method for handling the concentrated L-lactic acid solution of the moisture evaporation that sees through liquid that makes separatory membrane 3 with distillation procedure.Wherein, during distillation, distill so that distill the moisture concentration of stoste and fix while preferably supply with moisture.The L-lactic acid aqueous solution concentrates by heating evaporation moisture after distillating, and can obtain the purifying L-lactic acid of purpose concentration.When the L-lactic acid aqueous solution that obtains to contain low boiling point components such as ethanol or acetic acid during as distillate, removing low boiling point component in L-lactic acid concentration process is preferred mode.Behind the distillation procedure, for distillate, as required, spent ion exchange resin, activated carbon and chromatographic separation etc. are removed impurity, can also obtain more highly purified L-lactic acid.
The same when making D-lactic acid, methods such as separation that sees through D-lactic acid contained in the liquid that obtains from above-mentioned separatory membrane 3 and purifying can knownly in the past concentrate by combination, distillation and partial crystallization are carried out.For example, can enumerate by with the seeing through below the pH regulator to 1 of liquid of separatory membrane 3, the method for extracting with diethyl ether or ethyl acetate etc. then; Be adsorbed in ion exchange resin, clean the method for back wash-out; In the presence of acid catalyst, make it and the alcohol reaction, as ester distillatory method; And crystallization is the method for calcium salt or lithium salts etc.Preferably, can enumerate the method that available distillation procedure is handled the concentrated D-lactic acid solution of the moisture evaporation that sees through liquid that makes separatory membrane 3.Wherein, during distillation, distill so that distill the moisture concentration of stoste and fix while preferably supply with moisture.The D-lactic acid aqueous solution concentrates by heating evaporation moisture after distillating, and can obtain the purifying D-lactic acid of purpose concentration.When the D-lactic acid aqueous solution that obtains to contain low boiling point component (ethanol, acetic acid etc.) during as distillate, removing low boiling point component in D-lactic acid concentration process is preferred mode.Behind the distillation procedure, for distillate, as required, spent ion exchange resin, activated carbon and chromatographic separation etc. are removed impurity, can also obtain more highly purified D-lactic acid.
Then, can be used to make alcoholic acid microorganism or culturing cell as of the present invention, be not particularly limited, but for example can use, belong to the yeast that yeast (Saccharomyces) belongs to, kluyveromyces (Kluyveromyces) belongs to or fission yeast bacterium (Schizosaccharomyces) belongs to.This wherein can use yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Kluyveromyces lactis (Kluyveromyces lactis), schizosaccharomyces pombe (Schizosaccharomyces pombe) suitably.And, can also preferably use the bacterium that belongs to lactobacillus (Lactobacillus) or zymomonas (Zymomonas) genus.This wherein can use short lactobacillus (Lactobacillus brevis), zymomonas mobilis (Zymomonas mobilis) suitably.
The microorganism that can use in alcoholic acid production or culturing cell can be microorganism or the culturing cells that has improved the alcohol production ability artificially.Also can be the microorganism or the culturing cell that cause part character to change owing to sudden change or gene recombination particularly.As the microorganism of part character change or an example of culturing cell, can enumerate and integrate the glucoamylase gene that belongs to the Rhizopus mould and the yeast (microorganism, 3:555-564 (1987)) that has obtained farinose assimilative capacity.
And the alcoholic acid contained the liquid that sees through that obtains from above-mentioned separatory membrane 3 separates and purifying, can for example use suitably, and the method for purification of being undertaken by distillation method, or use the concentrating of separatory membrane, the method for purification of NF, RO film or zeolite system.
As the microorganism or the culturing cell that can in the production of pyruvic acid of the present invention, use, be not particularly limited, preferably for example can use, belong to the bacterium that pseudomonas (Pseudomonas) belongs to, excellent bacillus (Corynebacterium) belongs to, Escherichia (Escherichia) belongs to or acinetobacter calcoaceticus (Acinetobacter) belongs to.More preferably can use Pseudomonas fluorescens (Pseudomonas fuluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), intestinal bacteria bacteriums such as (Escherichia coli).
The microorganism or the culturing cell that can be used for pyruvic acid production can be microorganism or the culturing cells that has artificially improved pyruvic acid throughput, also can use the microorganism or the culturing cell that cause part character to change owing to sudden change or gene recombination.For example, can also preferably use sudden change or lacked the bacterium of participating in the ATPase gene that ATP produces by oxidative phosphorylation directly.And, can also preferably use mould, yeast etc.For example, can use mould or the yeast that belongs to yeast (Saccharomyces) genus, torulopsis (Torulopsis), mycocandida (Candida), Schizophyllum (Schizophyllum).More preferably, can use yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces copsis, Candida glabrata (Candida glabrata), Candida lipolytica (Candida lipolytica), torulopsis glabrata (Torulopsis glabrata), Split-gill moulds such as (Schizophyllum commune) or yeast to prepare pyruvic acid.
And the separation and the purifying that see through pyruvic acid contained the liquid from above-mentioned separatory membrane 3 obtains can be undertaken by the method for using anion-exchange column.For example, can use the spy to open the method for purification of disclosed employing weakly alkaline ion exchanger among the flat 6-345683 suitably.
As the microorganism or the culturing cell that can in the production of succsinic acid of the present invention, use, can use suitably to belong to the bacterium that anaerobism spirillum (Anaerobiospirillum) belongs to or actinobacillus (Actinobacillus) belongs to.Particularly, can enumerate the Anaerobiospirillum succinoproducens (Anaerobiospirillum succiniciproducens) put down in writing in No. 5143833 specification sheets of United States Patent (USP) or by the disclosed succsinic acid actinobacillus of James B.Mckinlay (Actinobacillus succinogenes) (Appl.Microbiol.Biotechnol., 71,6651-6656 (2005).And, can also use excellent bacillus (Corynebacterium) to belong to or rod-like stem bacterial type bacterium (Coryneform bacterium) such as tyrothricin (Brevibacterium) genus and intestinal bacteria etc.In the rod-like stem bacterial type bacterium, suitable is Corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium flavum (Brevibacterium flavum) and brevibacterium (Brevibacterium lactofermentum) etc.
The microorganism or the culturing cell that can be used for production of succinic acid can be microorganism or the culturing cells that has improved the alcohol production ability artificially.As object lesson, can use the microorganism that causes the throughput of succsinic acid to be improved by gene recombination, also can make the production of succinic acid raising thus.As this microorganism, for example can use special opened the disappearance put down in writing in the 2005-27533 communique pyruvate formate-lyase (pyruvate formate lyase) put down in writing in No. 5770435 specification sheets of Corynebacterium glutamicum (Corynebacterium glutamicum), United States Patent (USP) of record in the brevibacterium flavum MJ233AB-41 of serum lactic dehydrogenase (preserving number FERM BP-1498), the above-mentioned non-patent literature 1 and the disappearance strain-intestinal bacteria AFP111 strain of serum lactic dehydrogenase etc.
And the separation and the purifying that see through the succsinic acid that contains in the liquid by above-mentioned separatory membrane 3 obtains can use conventional succsinic acid method of purification.For example, can use the spy to open the disclosed method of purification that has made up water decomposition electrodialysis process and concentrating under reduced pressure, partial crystallization in the 2005-333886 communique suitably.
Microorganism or culturing cell as using in the production of methylene-succinic acid of the present invention are not particularly limited, and as object lesson, can preferably use mould or yeast.More preferably can enumerate the yeast that adopts the mould that belongs to Aspergillus (Aspergillus) or Ustilago (Ustilago) and belong to mycocandida (Candida), Rhodotorula (Rhodotorula) and carry out the production of methylene-succinic acid.This wherein preferably uses terreus (Aspergillus terreus), the mould of separating equisetic acid aspergillus (Aspergillus itaconicus), Ustilago maydis (D C.) Corola. (Ustilago maydis), Bermuda grass ustilago (Ustilago cynodontis) and Rabin ustilago (Ustilago rabenhorstina) or antarctic candida (Candia antarctica) in the production of methylene-succinic acid.
And the separation and the purifying that see through the methylene-succinic acid that contains in the liquid that are obtained by above-mentioned separatory membrane 3 preferably can adopt ultrafiltration or electrodialysis to carry out.For example, can use disclosed method of purification of being undertaken in special public clear-No. 50958 communiques suitably by the electrodialysis of adopting ultrafiltration or salt type cation exchange resin membrane.
As can be of the present invention 1, microorganism of using in the production of ammediol or culturing cell, be not particularly limited, as object lesson, can enumerate, in the wild-type strain, have by glycerine and synthesize 1, the microorganism that belongs to klebsiella (Klebsiella) genus, clostridium (Clostridium) genus, Bacterium lacticum (Lactobacillus) genus of the ability of ammediol.
Making 1 by glycerine, during ammediol, microorganism preferably contains at least one gene that (a) coding has the active polypeptide of glycerol dehydratase; (b) at least one gene of the encoding glycerol dehydratase sensitization factor again; (c) coding has and converts the 3-hydroxy propanal to 1, at least one gene of the non-specific catalyst activity of ammediol.
More preferably using can be by glucose production 1, the recombinant microorganism of ammediol.Host as recombinant microorganism, be preferably and be selected from Klebsiella (Klebsiella), fusobacterium (Clostridium), lactobacillus (Lactobacillus), Citrobacter (Cytrobacter), enterobacter (Enterobacter), aerobacter (Aerobacter), Aspergillus (Aspergillus), yeast belong (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), zygosaccharomyces belongs to (Zygosaccharomyces), Pichia (Pichia), genus kluyveromyces (Kluyveromyces), mycocandida (Candida), Hansenula (Hansenula), Debaryomyces (Debaryomyces), Mucor (Mucor), torulopsis (Torulopsis), the methyl bacterium belongs to (Methylobacter), salmonella (Salmonella), bacillus (Bacillus), aerobacter (Aerobacter), streptomyces (Streptomyces), recombinant microorganism in Escherichia (Eschericia) and the Rhodopseudomonas (Pseudomonas), more preferably intestinal bacteria.
Can be by glucose production 1, the recombinant microorganism of ammediol is preferably and for example contains at least one gene that (a) coding has the active polypeptide of glycerol-3-phosphate dehydrogenase; (b) coding has the recombinant microorganism of at least one gene of the polypeptide of glycerol-3-phosphate enzymic activity.And then, more preferably contain the glycerol dehydratase sensitization factor again and be recombinant microorganism by the gene of the coding of isolating orfX and orfZ from the dha regulon.And then more preferably, recombinant microorganism is for having lacked glycerol kinase activity and/or glycerol dehydrogenase enzymic activity and/or the active recombinant microorganism of triosephosphate isomerase.
And, by seeing through of obtaining of above-mentioned separatory membrane 3 contain in the liquid 1, the separation of ammediol and purifying can by concentrate, partial crystallization carry out.For example, can use the method for purification of disclosed employing concentrating under reduced pressure, partial crystallization in Te Kaiping-No. 35785 communique suitably.
As the microorganism or the culturing cell that can in the production of cadaverine of the present invention, use, be not particularly limited, but as object lesson, the lysine decarboxylase and/or the proteic enzymic activity enhanced of the Methionin cadaverine antiport microorganism that have of Institute of Micro-biology preferably.More preferably, can enumerate the recombinant microorganism of having integrated coding lysine decarboxylase and/or the proteic gene of Methionin cadaverine antiport.More preferably, can enumerate the microorganism of the gene of having integrated the coding lysine decarboxylase more than a kind or 2 kinds.
When making cadaverine with recombinant microorganism, preferably, more preferably has the rod-like stem bacterial type bacterium that lysine decarboxylase is active and have any feature at least in homoserine auxotroph or S-(2-the aminoethyl)-L-halfcystine patience with intestinal bacteria or rod-like stem bacterial type bacterium recombinant microorganism as the host.And, in the rod-like stem bacterial type bacterium, more preferably be selected from Corynebacterium or brevibacterium sp, more preferably Corynebacterium glutamicum (Corynebacuterium gulutamicum).Further, preferred microorganism disappearance homoserine dehydrogenase activity more preferably causes lacking homoserine dehydrogenase activity owing to gene inserts the generation that suddenlys change.
And known in the past methods such as separation that sees through the cadaverine that contains in the liquid and the purifying that is obtained by above-mentioned separatory membrane 3 can concentrate by having made up, distillation and partial crystallization are carried out.For example, can use the spy to open the method for purification of disclosed employing partial crystallization in the 2004-222569 communique suitably.Among the present invention, various polymer raw materials are made in spendable acid in the time of can be by cultured continuously, in needing the polymer raw material purposes of good purity, and the preferred purification process that is undertaken by partial crystallization that uses.If keep the pH of nutrient solution, then can reclaim the cadaverine dihydrochloride by partial crystallization from aforementioned seeing through the liquid with hydrochloric acid.More preferably, keep the pH of nutrient solution in the time of can being set forth in cultured continuously with dicarboxylic acid, reclaim the cadaverine dicarboxylate.At this moment, dicarboxylic acid preferably functional group has only the aliphatics and/or the aromatic dicarboxylic acid of 2 carboxyls, is more preferably hexanodioic acid, sebacic acid, 1, arbitrary in 12-dodecanedicarboxylic acid, succsinic acid, m-phthalic acid or the terephthalic acid.
As the microorganism or the culturing cell that can in the production of nucleic acid of the present invention, use, be not particularly limited, can be throughput from the isolating original nucleic acid of nature with regard to high microorganism or culturing cell, also can be the prokaryotic micro-organisms that has improved throughput artificially.Particularly, can be to cause reformed microorganism of part character or culturing cell owing to sudden change or gene recombination.
This wherein describes for the change of aforementioned part character.In order effectively to produce nucleic acid, need biosynthesizing nucleic acid and accumulation, be discharged into outside the organism.Therefore, by the enzyme that strengthens the biosynthetic pathway that participates in nucleic acid, the enzymic activity that reduces the decomposition approach that participates in nucleic acid, and change and nucleic acid are discharged into that the outer relevant protein of organism or organism film are formed or the like, the character of change microorganism or culturing cell, can effectively prepare microorganism or the culturing cell of producing nucleic acid.
Particularly, when producing Trophicardyl, it is desirable to microorganism or culturing cell and do not have adenylosuccinic acid synthase activity or faint.And, it is desirable to not have hypoxanthylic acid dehydrogenase activity or faint.And, it is desirable to not have nucleosidase activity or faint.When producing guanosine, it is desirable to microorganism or culturing cell and do not have adenylosuccinic acid synthase activity or faint.And, it is desirable to not have guanylic reductase activity or faint.And, it is desirable to not have nucleosidase activity or faint.And, it is desirable to not have activity of 5 '-nucleotidase or faint.When producing uridine, it is desirable to microorganism or culturing cell and do not have Uridine phosphorylase activity or faint.When producing cytidine, it is desirable to not have cytidine deaminase activity or faint, it is desirable to not have homoserine dehydrogenase activity or faint.
As the microorganism that can when the present invention makes nucleic acid, use or culturing cell, can preferably use rod-like stem bacterial type bacterium or withered grass bacterium.For example, when producing Trophicardyl, as rod-like stem bacterial type bacterium, can enumerate the bacterium that belongs to Corynebacterium (Genus Corynebacterium), preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), product ammonia rod bacillus (Corynebacterium ammoniagenes), Corynebacterium guanofaciens or the Corynebacterium Petrophilium of using in Corynebacterium.In addition, as the withered grass bacterium, for example can enumerate, belong to the bacterium of bacillus (Bacillus), wherein preferably use subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus liqueniformis), bacillus pumilus (Bacillus pumilus).And, when producing guanosine, as rod-like stem bacterial type bacterium, can enumerate and belong to the bacterium that excellent bacillus (Corynebacterium) belongs to, this wherein preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), as the withered grass bacterium, can enumerate the bacterium that for example belongs to genus bacillus (Bacillus) genus, this wherein preferably uses subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus liqueniformis) or bacillus pumilus (Bacillus pumilus).In addition, when producing uridine or cytidine, as the withered grass bacterium, this wherein can preferably use and belong to the bacterium that genus bacillus (Bacillus) belongs to, and wherein preferably uses subtilis (Bacillus subtilis).
And the separation and the purifying that see through the nucleic acid that contains in the liquid by above-mentioned separatory membrane 3 obtains are preferably undertaken by having made up ion exchange resin treatment method, concentrated cooling crystallization, membrane separation process and other method.In order to remove impurity, can use the active carbon adsorption of ordinary method and recrystallization method to carry out purifying.
When the present invention makes amino acid, as this amino acid, can enumerate preferred L-Threonine, L-Methionin, L-L-glutamic acid, L-tryptophane, L-Isoleucine, L-glutaminate, L-arginine, L-L-Ala, L-Histidine, L-proline(Pro), L-phenylalanine, L-aspartic acid, L-tyrosine, methionine(Met), Serine, Xie Ansuan, leucine.
Microorganism or culturing cell when for example making the L-Threonine can use the bacterium that belongs in Escherichia (Escherichia), Providencia (Providencia), Corynebacterium (Corynebacterium), brevibacterium sp (Brevibacterium) or the serratia (Serratia).This wherein, particularly preferred bacterium is intestinal bacteria (Escherichia coli), providencia rettgeri (Providencia rettgeri), Corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium (Brevibacterium lactofermentum) or serratia marcescens (Serratia marcescens).
As the microorganism or the culturing cell that can when making L-Methionin or L-L-glutamic acid, use, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum) or brevibacterium (Brevibacterium lactofermentum).
As microorganism or the culturing cell when making the L-tryptophane, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium (Brevibacterium lactofermentum), subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or intestinal bacteria (Escherichia coli).
Microorganism or culturing cell during as manufacturing L-Isoleucine, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium (Brevibacterium lactofermentum) or serratia marcescens (Serratia marcescens).
Microorganism or culturing cell when making L-glutaminate, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium (Brevibacterium lactofermentum) or in add Flavobacterium (Flavobacterium rigense).
Microorganism or culturing cell during as manufacturing L-arginine, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), serratia marcescens (Serratia marcescens), intestinal bacteria (Escherichia coli) or subtilis (Bacillus subtilis).
Microorganism or culturing cell during as manufacturing L-L-Ala, preferred brevibacterium flavum (Brevibacterium flavum) or oxidation Arthrobacter (Arthrobacter oxydans)
Microorganism or culturing cell during as manufacturing L-Histidine, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), Brevibacterium ammoniagenes (Brevibacterium ammoniagenes), serratia marcescens (Serratia marcescens), intestinal bacteria (Escherichia coli), subtilis (Bacillus subtilis) or streptomyces coelicolor (Streptomyces coelicolor).
Microorganism or culturing cell during as manufacturing L-proline(Pro), preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), Kurthia catenaforma, serratia marcescens (Serratia marcescens) or intestinal bacteria (Escherichia coli).
Microorganism or culturing cell during as manufacturing L-phenylalanine or L-tyrosine, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium (Brevibacterium lactofermentum) or intestinal bacteria (Escherichia coli).
Microorganism or culturing cell during as manufacturing L-aspartic acid, preferred brevibacterium flavum (Brevibacterium flavum), Bacillus megatherium (Bacillus megatherium), intestinal bacteria (Escherichia coli) or Pseudomonas fluorescens (Pseudomonas fluorescens).
Microorganism or culturing cell during as the manufacturing methionine(Met), preferred Corynebacterium glutamicum (Corynebacterium gulutamicum).
Microorganism or culturing cell during as the manufacturing Serine, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium (Brevibacterium lactofermentum) or oxidation Arthrobacter (Arthrobacter oxydans).
Microorganism or culturing cell during as the manufacturing Xie Ansuan, preferred brevibacterium (Brevibacterium lactofermentum), serratia marcescens (Serratia marcescens) or Klebsiella pneumonia (Klebsiella pneumoniae).
Microorganism or culturing cell during as the manufacturing leucine, preferred Corynebacterium glutamicum (Corynebacterium gulutamicum), brevibacterium (Brevibacterium lactofermentum) or serratia marcescens (Serratia marcescens).
Aforesaidly can be used to make amino acid whose microorganism or culturing cell, can separate originally amino acid throughput with regard to high microorganism or culturing cell from occurring in nature, also can be microorganism or the culturing cell that illustrative microorganism or culturing cell has artificially been improved its throughput.In addition, can also be microorganism or the culturing cell that causes part character to change owing to sudden change or gene recombination.
The Corynebacterium glutamicum (Corynebacterium gulutamicum) that the L-L-Ala productivity put down in writing among providencia rettgeri (Providencia rettgeri) that a microorganism that changes as part character or an example of culturing cell, the L-Threonine productivity that has the spy to open to put down in writing among the flat 2-219582 have improved and the flat 3-500486 of special table has improved etc.
Below, describe can be used as the porous membrane that separatory membrane preferably uses among the present invention.
As porous membrane, can use with organic materialss such as inorganic materials such as pottery, resins as raw-material porous membrane, but more preferably use the porous separatory membrane that contains porous matter resin layer.This porous membrane has had the porous matter resin layer of separation work ergosphere effect on the surface of porous matter base material.Porous matter base material is supported porous matter resin layer, gives separatory membrane intensity.Porous matter resin layer can be soaked in the porous matter base material, also can not be soaked in the porous matter base material, but consider from the intensity aspect, the preferred film that is soaked in the porous matter base material that adopts.
The material of porous matter base material is made of organic materials and/or inorganic materials etc., the wherein desirable organic fibre that uses.Preferred porous matter base material is to use weaving cotton cloth of organic fibres formations such as cellulosic fibre, tri acetic acid fiber cellulose fiber, trevira, polypropylene fibre and polyethylene fibre or non-woven fabrics etc.Wherein preferably use the density control ratio to be easier to, to make and also be easy to cheap non-woven fabrics.
Porous matter resin layer as mentioned above, plays the separation work ergosphere, can use organic high molecular layer suitably.Material as organic high molecular layer, for example can enumerate polythylene resin, polypropylene-based resin, polyvinyl chloride resin, polyvinylidene fluoride vinyl resin, polysulfones resin, polyether sulfone resin, polyacrylonitrile resinoid, polyolefin resin, cellulosic resin and cellulose triacetate resinoid etc.Organic high molecular layer can be with the resin compound of these resins as main component.This wherein as main component, is meant and contains this composition that 50 weight % are above, preferred 60 weight % are above.Wherein, as the pleurodiaphragmatic in terspace material that constitutes porous matter resin layer, be to be easy to and the weather resistance of physics and also excellent polyvinyl chloride resin, polyvinylidene fluoride vinyl resin, polysulfones resin, polyether sulfone resin, polyacrylonitrile resinoid or the polyolefin resin of resistance preferably by solution system film, more preferably polyvinylidene fluoride vinyl resin or polyolefin resin, most preferably polyvinylidene fluoride vinyl resin or with its resin as main component.
Here, as the polyvinylidene fluoride vinyl resin, preferably use vinylidene homopolymer or with can with the interpolymer of the vinyl monomer of vinylidene copolymerization.As can with the vinyl monomer of vinylidene copolymerization, can enumerate tetrafluoroethylene, R 1216 and trichloro-monofluoro ethene etc.In addition,, can enumerate polyethylene, polypropylene, polyvinyl chloride or polychloroprene as polyolefin resin, but preferred polyvinyl chloride.
Below, the summary of the making method of illustration porous membrane, and describe.
At first, the summary to the making method of the flat film in the porous membrane describes.Flat film obtains in the following manner, promptly on the surface of porous matter base material, form the tunicle of the system pleurodiaphragmatic in terspace liquid contain the resin that constitutes porous matter resin layer and solvent, this system pleurodiaphragmatic in terspace liquid be impregnated in the porous matter base material, then, the tunicle side surface of the porous matter base material with tunicle is contacted with the precipitation bath that contains non-solvent, make hardening of resin, on the surface of porous matter base material, form porous matter resin layer simultaneously.At this moment, although can select according to purposes, but the mean thickness of porous matter base material is preferably below the above 3000 μ m of 50 μ m, and the mean thickness of porous matter resin layer is below 5000 μ m more than the 20 μ m, more preferably the following scope of the above 2000 μ m of 50 μ m.
Below, the summary of the manufacture method of hollow-fibre membrane is described.Hollow-fibre membrane can spue by the pipe from the double tube type metal mouth outside and contain the system pleurodiaphragmatic in terspace liquid that is made of resin that constitutes porous matter resin layer and solvent, form and use fluid from the pipe of the double tube type metal mouth inboard hollow bulb that spues simultaneously, the mode of cooling curing is made in cooling bath then.At this moment, the internal diameter of tubular fibre is the scope below 5000 μ m more than the 200 μ m preferably, and the thickness of porous matter resin layer is the scope below 2000 μ m more than the 20 μ m preferably.In addition, can also contain fabric or the volume thing of making tubular by organic fibre or inorganic fibre in the inside of tubular fibre.
On the outside surface of the hollow-fibre membrane that obtains, can also apply (stacked) new porous resin layer.The stacked of this porous matter resin layer can for example wetting ability or hydrophobicity, fine pore etc. be varied to desirable character and carry out for the character that makes hollow-fibre membrane.
This stacked from the teeth outwards porous matter resin layer can be made in the following manner, promptly by the stoste of resin dissolves in solvent is contacted with the precipitation bath that contains non-solvent, makes hardening of resin.The material of stacked resin for example can preferably be used the same material of material with above-mentioned porous matter resin layer.In addition, laminating method is not particularly limited, hollow-fibre membrane can be impregnated in the stoste, also can on the surface of hollow-fibre membrane, be coated with stoste, stacked after, by extruding the part of the stoste of paying, blow away with air knife, regulate lamination amount.
The preferred 0.01 μ m of the average fine pore of the porous membrane that uses among the present invention is above less than 1 μ m.If the average fine pore of porous membrane 0.01 μ m is above and less than 1 μ m, has the obstruction that the thalline that uses when being not easy to take place by fermentation causes, and keep the performance of strainability long-term and stably.And, if the average fine pore of porous membrane can realize not spilling the high elimination factor of microorganism or culturing cell more than 0.01 μ m and less than 1 μ m, and realize keeping for a long time high water-permeability.
If near the size of microorganism or culturing cell, owing to have the situation of microorganism or the direct plugging hole of culturing cell, so the average fine pore of porous membrane is preferably less than 1 μ m.In addition, the average fine pore of porous membrane in order to prevent the spilling of microorganism or culturing cell, to be the generation of this trouble that reduces of elimination factor, is preferably compared with the size of microorganism or culturing cell, not aperture that can be excessive.Therefore, in microorganism or the culturing cell, when using little yeast of cell or bacterium etc., average fine pore is more preferably below 0.4 μ m, more preferably below 0.2 μ m.In addition, there is microorganism or culturing cell production purpose chemical material in addition, the easy situation of agglutinative material such as protein, polyose for example, but also exist because the part death of microorganism in the nutrient solution or culturing cell causes the situation of celliferous broken thing.The obstruction of the porous membrane that causes for fear of these materials, average fine pore more suitably is below 0.1 μ m.
As seen from the above, the average fine pore of porous membrane of the present invention is preferably below 0.4 μ m, more preferably below 0.2 μ m, most preferably below 0.1 μ m.
On the one hand, if average fine pore is too small, the water permeability of porous membrane then reduces, even film is not contaminated, also can't effectively turn round, so among the present invention the average fine pore of porous membrane preferably more than 0.01 μ m.More than 0.02 μ m, more suitably be more than the 0.04 μ m more preferably.
Here, average fine pore can be observed down by the scanning electron microscope that is determined at 10,000 times of multiplying powers, and diameter that can observed all pores in the scope of 9.2 μ m * 10.4 μ m is tried to achieve after averaging.And, at pore not during Cheng Yuanzhuan,, obtain and the area of pore circle of the same area (circle of equal value), with the diameter of circular diameter of equal value as pore with image processing apparatus etc.
The standard deviation of the fine pore of operable separatory membrane is more little among the present invention, i.e. the distribution of fine pore size is narrow more, and is then good more.The narrowly distributing of the size of preferred fine pore, standard deviation is below 0.1 μ m.The standard deviation of fine pore is little, and promptly fine pore is of the same size, can obtain the liquid that sees through of homogeneous characteristic, and the running management of device has become easily also.
The standard deviation of fine pore can calculate by following (formula 5), in this formula with can observed pore number in above-mentioned 9.2 μ m * 10.4 mu m ranges as N, with each diameter of measuring as X k, with the mean value of pore diameter as X (ave).
[several 5]
σ = Σ k = 1 N ( X k - X ( ave ) ) 2 N .... (formula 5)
In the separatory membrane that the present invention uses, the perviousness that contains the nutrient solution of chemical is one of important performance, can adopt the index of the pure water transmission coefficient of the separatory membrane before using as perviousness.Among the present invention, the pure water transmission coefficient of separatory membrane uses the purified water through 25 ℃ temperature of reverse osmosis membrane processing, when measuring permeable amount and calculate with drop height 1m, is preferably 1 * 10 -10m 3/ m 2More than/the s/Pa.And in order to obtain practical enough liquid measures that sees through that, preferred pure water transmission coefficient is 2 * 10 - 9m 3/ m 2/ s/Pa above 6 * 10 -7m 3/ m 2Below/the s/Pa, more preferably 2 * 10 -9m 3/ m 2/ s/Pa above 2 * 10 -7m 3/ m 2Below/the s/Pa.
The film surfaceness of the separatory membrane that uses among the present invention is to influence the factor that separatory membrane stops up.For coefficient of overburden and the film opposing that makes separatory membrane suitably reduces, carry out the manufacturing of chemical with lower intermembranous differential pressure, the film surfaceness of separatory membrane is preferably below 0.1 μ m.Owing to, can realize manufacturing chemistry product stably, so the preferred surface roughness is the smaller the better by suppressing obstruction.
And the film surfaceness is in order to make the microorganism or the culturing cell that are attached to separation membrane surface flow the face cleaning performance that forms and one of the factor of peeling off easily owing to stirring or by the liquid that circulation tube produces easily.According to this viewpoint, the film surfaceness of preferable separation film is the smaller the better, preferably below 0.1 μ m.If microorganism or culturing cell that surfaceness below 0.1 μ m, is attached on the film are just peeled off easily.
In addition, reach below the 0.1 μ m, in the filtration of microorganism or culturing cell, the shearing force that takes place on the film surface is reduced, suppress destruction microorganism or culturing cell by film surfaceness with porous membrane.Consequently, because the obstruction of separatory membrane also is suppressed, therefore can realize filtration steady in a long-term.
Here, so-called film surfaceness, be and the surperficial mean value that changes of the film of face direction vertical direction, as described below, can use following atomic force microscope device (AFM) to measure.
Device: atomic force microscope device (Digital Instruments (strain) system Nanoscope IIIa)
Condition
Probe: SiN cantilever (Digital Instruments (strain) system)
Scan pattern contact mode (measuring in the gas)
Rap (measuring in the water) in the water
Sweep limit 10 μ m, 25 μ m four directions (measuring in the gas)
5 μ m, 10 μ m four directions (measuring in the water)
Scanning resolution 512 * 512
Specimen preparation: survey periodic membrane sample, be immersed under the normal temperature in the ethanol after 15 minutes, after impregnated in the RO water 24 hours and cleaning, air-dryly use again.So-called RO water is that a kind of filter membrane of employing is that reverse osmosis membrane (RO film) filters, and has got rid of the water of the impurity of ion and salt etc.The size in the hole of RO film is probably below 2nm.
The height of the Z-direction of the each point that obtains according to above-mentioned AFM is with following (formula 6) calculating film surfaceness d Rough
[several 6]
d rough = Σ n = 1 N | Z n - Z ‾ | N .... (formula 6)
D Rough: surfaceness (μ m)
Z n: the height of Z-direction (μ m)
: the center line average of sweep limit (μ m)
N: working sample number
Above-mentioned this separatory membrane can cooperate the shape of membrane sepn groove, suitably machining shape.For example, the separatory membrane of flat film form can make up by the support with other preparation and make this separating film element shown in Figure 3.In addition, by adhere, seal the hollow bulb of hollow-fibre membrane with materials such as resins, can make separating film element shown in Figure 4.In addition, among the present invention, consider, preferably use hollow-fibre membrane from the favourable this viewpoint of being provided with of the membrane area of unit volume.
Below, adopt accompanying drawing, the summary of these separating film elements is described.
Fig. 3 is the summary oblique drawing of an embodiment that is used to illustrate the separating film element of the separatory membrane that adopts flat film form.Separating film element, as shown in Figure 3, by configuration flow pipeline material 19 and separatory membrane 20 and constitute successively on two sides with inflexible supporting plate 18.Supporting plate 18 has recess 21 on its two sides.Separatory membrane 20 filters nutrient solution.Flow channel material 19 is to make through the separatory membrane 20 filtering materials that effectively flow to supporting plate 18 through liquid.Flow to the recess 21 that liquid passes through supporting plate 18 that sees through that contains the production thing of supporting plate 18, the collector tube 22 through as device for transferring is fetched to the bactogen outside.Here, can use with the water level differential pressure to be representative pump, the suction strainer that is undertaken by liquid or gas etc. or to method such as pressurizeing in the apparatus system, as taking out the power that sees through liquid.
And, when cooperating culture tank to increase membrane area, can increase membrane area by stacked this separating film element.
Fig. 4 is the summary oblique drawing that is used to illustrate the separating film element of the separatory membrane that uses the tubular fibre form, and it is mainly by supporting plate 18, the separatory membrane 20 of tubular fibre form, and the resin-sealed layer 24 in upper resin sealing ply 23 and bottom constitutes.Separatory membrane 20 is supported to pencil by the resin-sealed layer 24 in upper resin sealing ply 23 and bottom and is adhered and be fixed on the supporting plate 18.Form the hollow bulb of the separatory membrane 20 of sealed hollow fibre shape by bottom resin-sealed layer 24 adhesion of carrying out and immobilization, prevented the structure that nutrient solution spills.On the other hand, the hollow bulb of the separatory membrane 20 of upper resin sealing ply 23 blow-by tubular fibre forms, hollow bulb is connected with collector tube 22.This separating film element can be arranged in the bactogen by supporting plate 18.By the separatory membrane 20 filtering hollow bulbs that see through liquid by hollow-fibre membrane,, be fetched to the bactogen outside through collector tube 22.As the power that is used to take out through liquid, the suction strainer that can use water level differential pressure, pump, is undertaken by liquid or gas etc. or to method such as pressurizeing in the apparatus system.
The membrane sepn groove 2 that possesses separatory membrane it is desirable to carry out high pressure steam sterilization, can avoid the pollution of assorted bacterium like this.So-called high pressure steam sterilization of the present invention is by steam, and the membrane sepn groove is heated and pressurizes, and makes to be present in microorganism or culturing cell deactivation in the groove.As heating and pressurized conditions, preference is heated more than 20 minutes under at 121.1 ℃, the condition of vapour pressure 1 air pressure.Therefore, the membrane sepn groove 12 of bactogen of the present invention and be configured in separatory membrane, element constituent material in this membrane sepn groove 12, the preferred material that uses with tolerance high pressure steam sterilization operation under this condition.Can comprise the interior sterilization of culture tank of separating film element thus.If can sterilize in the culture tank, so just can avoid the not danger of preferred microbial contamination of quilt when cultured continuously, can realize the stable continuous cultivation.
Constitute materials such as the separatory membrane of separating film element and supporting plate and preferably under such condition patience is being arranged more than 20 minutes under the condition of promptly 121.1 ℃ of the conditions of high pressure steam sterilization operation, vapour pressure 1 air pressure, so long as like this, then the kind to separatory membrane and element constituent material then is not particularly limited.As the starting material of separatory membrane, can adopt the starting material of above-mentioned porous membrane with this patience.In addition, as element constituent materials such as supporting plates, can preferably select for example resin such as metal such as stainless steel or aluminium or polyamide-based resin, fluorine-type resin, polycarbonate resin, polyacetals resin, polybutylene terephthalate resinoid, PVDF, modified polyphenyl ether resin and polysulfones resin.
Embodiment
Below, just the present invention enumerates embodiment, comparative example, is described in detail.
Particularly, in embodiment 1～9, comparative example 1～4, to selecting L-lactic acid as chemical, yeast (reference example 1) with L-lactic acid-producing ability is as microorganism or culturing cell, porous membrane (reference example 2: flat film) as separatory membrane, use the manufacturing of the successive chemical that the bactogen of Fig. 2,7,9,13～16 shown in arbitrary carry out, describe.
In addition, in embodiment 10, comparative example 5, to selecting cadaverine (1, the 5-pentamethylene diamine) as chemical, microorganism with cadaverine throughput is as microorganism or culturing cell, porous membrane (reference example 2:, use the manufacturing of the successive chemical that bactogen shown in Figure 2 carries out to describe flat film) as separatory membrane.
And, in embodiment 11, comparative example 6, to selecting L-Methionin as chemical, microorganism with L-lysine production ability is as microorganism or culturing cell, porous membrane (reference example 2:, use the manufacturing of the successive chemical that bactogen shown in Figure 2 carries out to describe flat film) as separatory membrane.
And, in all embodiment, adopt butterfly valve as flow rate control device 25, regulate the flow of the nutrient solution that flows to the membrane sepn groove thus and flow into and press.
And these embodiment are the explanations to a scheme of the present invention, do not limit the present invention.
(reference example 1) has the making of the yeast strains (SU014 strain) of lactic acid-producing ability
In the present embodiment,, use the yeast of the L-ldh gene importing in Africa xenopus (Xenopus laevis) source in PDC1 promotor downstream with sequence number 1 described base sequence as yeast with lactic acid-producing ability.The clone of the L-ldh gene in Africa xenopus source is undertaken by the PCR method.Among the PCR, with according to the phagemid dna of the incidental operational manual preparation in the cDNA library (STRATAGENE corporate system) in the kidney of Africa xenopus source as template.
Use KOD-Plus polysaccharase (corporate system is spun by Japan) in the pcr amplification reaction, reaction buffer, dNTPmix etc. adopt the incidental product of test kit.The reaction system for preparing 50 μ l, wherein the phagemid dna for preparing according to incidental working specification as mentioned above is that 50ng/ sample, primer are that 50pmol/ sample and KOD-Plus polysaccharase are 1 unit/sample.With pcr amplification device iCycler (BIO-RAD corporate system), make reaction soln 94 ℃ of temperature thermally denatures after 5 minutes, with 94 ℃ (thermally denatures) 30 seconds, 55 ℃ (annealing of primer) 30 seconds, 68 ℃ (complementary strand extensions) was a circulation in 1 minute, carried out 30 circulations, was cooled to 4 ℃ then.And primer set for amplification (sequence number 2,3) adds the SalI recognition sequence at 5 end sides, 3 end sides add the NotI recognition sequence and prepare.
The purifying pcr amplified fragment, is connected in the pUC118 carrier (cut off with Restriction Enzyme HincII, cut surface is carried out the carrier that dephosphorylation has been handled) after the terminal phosphateization with T4 polynucleotide kinase (TakaraBio corporate system).Connect with dna ligation kit Ver.2 (TakaraBio corporate system).Competent cell (TakaraBio corporate system) with connecting solution transformed into escherichia coli DH5 α is inoculated on the LB plate that contains 50 μ g/mL microbiotic penbritins, cultivates an evening.To the bacterium colony of growth, reclaim plasmid DNA with small-sized preparation method, cut off with Restriction Enzyme SalI and NotI, select the plasmid of the ldh gene that has inserted the Africa xenopus source.These a series of operations are all carried out according to incidental working specification.
Cut off the PUC118 carrier of the L-ldh gene that has inserted above-mentioned Africa xenopus source with Restriction Enzyme SalI and NotI,, contain the fragment of the L-ldh gene in Africa xenopus source according to the ordinary method purifying by 1% agarose gel electrophoresis DNA isolation fragment.The fragment that contains the L-ldh gene that obtains is connected in the XhoI/NotI place of incision of expression vector pTRS11 shown in Figure 5, reclaim plasmid DNA with method same as described above, by cut off, select the ldh expression carrier of having inserted the Africa xenopus source with Restriction Enzyme XhoI and NotI.After, with the integration made like this L-ldh expression carrier in Africa xenopus source be called pTRS102.
With this pTRS102 as amplification template, by with oligonucleotide (sequence number 4,5) as the PCR of primer sets, amplify the PCR fragment of the 1.3kb of the L-ldh gene that contains Africa xenopus source and TDH3 terminator sequence.Here, sequence number 4 is designed to add the sequence corresponding to the upstream from start codon 60bp of PDC1 gene.
Then, with plasmid pRS424 as amplification template, by with oligonucleotide (sequence number 6,7) as the PCR of primer sets, amplify the PCR fragment that contains as the 1.2kb of the TRP1 gene of yeast selective marker.Here, sequence number 7 is designed to add the sequence corresponding to PDC1 gene terminator codon downstream 60bp.
Separate each dna fragmentation, purifying according to a conventional method with 1% agarose gel electrophoresis.To have mixed each the 1.3kb fragment that obtains, the segmental mixture of 1.2kb herein as amplification template, by with oligonucleotide (sequence number 4,7), amplified the PCR fragment of having added corresponding to being connected of the sequence of the upstream and downstream 60bp of PDC1 gene about 2.5kb of L-ldh gene, TDH3 terminator and TRP1 gene that Africa xenopus originates at 5 terminal and 3 ends respectively as the PCR method of primer sets.
Separate above-mentioned PCR fragment with 1% agarose gel electrophoresis, according to a conventional method behind the purifying, yeast saccharomyces cerevisiae in the transformed yeast (Saccharomyces cerevisae) NBRC10505 strain, cultivate with the non-interpolation substratum of tryptophane, the L-ldh gene of selecting the Africa xenopus source imports the transformant in the PDC1 gene promoter downstream on karyomit(e).
Is that L-ldh gene importing yeast in PDC1 gene promoter downstream on karyomit(e) that Africa xenopus is originated is confirmed by following mode to the transformant that obtains with aforesaid way.At first, genomic dna with genome DNA extracting reagent kit " Gen と Ru く ん " (registered trademark) (Takarabio corporate system) preparation transformant, with it as amplification template, by with oligonucleotide (sequence number 8,9), confirm to have obtained the amplification of DNA fragments of about 2.8kb as the PCR of primer sets.And, in the non-transformant, obtained the amplification of DNA fragments of about 2.1kb by above-mentioned PCR.Below, the transformant that the L-ldh gene in above-mentioned Africa xenopus source is imported the PDC1 gene promoter downstream on karyomit(e) is called the B2 strain.And, the upstream and downstream sequence of PDC1 gene can from the yeast belong genome database ( URL:http: //www.yeastgenome.org/) obtain.
Then, making the pdc1 gene substitution of putting down in writing in the international open WO2007/097260 brochure is the TRP1 mark, and the yeast SW015 strain that has the thermal sensibility variation in the pdc5 gene engages with the B2 strain of above-mentioned acquisition, obtains 2 times of somatocyte.These 2 times of somatocyte are formed in the substratum ascus form ascus.Dissect ascus with micromanipulator, obtain each haploid cell, study the auxotroph of each haploid cell.From the haploid cell that obtains, select the ldh gene that in the pdc1 locus, has inserted Africa xenopus (Xenopus laevis) source, and have (can not grow for 34 ℃) strain of thermal sensibility variation in the pdc5 gene.The yeast strains that obtains is called the SU014 strain.
And, measure SC substratum (METHODSIN YEAST GENETICS 2000 EDITION by the HPLC method under the condition shown in following, CSHL PRESS) cultivated contained lactic acid in the culture supernatant of transformant in, thereby confirmed whether the SU014 strain has the lactic acid-producing ability.
Post: Shim-Pack SPR-H (island Jin She system)
Mobile phase: 5mM tosic acid (flow velocity 0.8mL/ minute)
Reaction solution: 5mM tosic acid, 20mM Bis-Tris, 0.1mM EDTA2Na (flow velocity 0.8mL/ minute)
Detection method: electroconductibility
Temperature: 45 ℃.
In addition, the optical purity of L-lactic acid is measured under the following conditions and is measured by the HPLC method.
Post: TSK-gel Enantio L1 (TOSOH Co., Ltd's system)
Mobile phase: 1mM copper sulfate solution
Flow velocity: 1.0ml/ minute
Detection method: UV254nm
Temperature: 30 ℃.
In addition, the optical purity of L-lactic acid is calculated with following formula.
Optical purity (%)=100 * (L-D)/(L+D)
Wherein, L represents the L-concentration of lactic acid, and D represents the D-concentration of lactic acid.
The result that HPLC analyzes is, detects L-lactic acid, and D-lactic acid is detecting below the boundary.According to above research, confirmed that this SU014 strain has L-lactic acid-producing ability.
The making of (reference example 2) flat film of porousness
Use polyvinylidene difluoride (PVDF) (PVDF) resin as resin respectively, and N,N-dimethylacetamide (DMAc) fully stirs them as solvent under 90 ℃ temperature, obtained to have the stoste of following composition.
PVDF:13.0 weight %
DMAc:87.0 weight %.
Then, above-mentioned stoste is after being cooled to 25 ℃ temperature, and being coated on the density that is attached in advance on the sheet glass is 0.48g/cm 3, thickness is on the trevira system non-woven fabrics (porous matter base material) of 220 μ m, impregnated in the precipitation bath with following composition of 25 ℃ of temperature 5 minutes immediately, obtained to have formed on porous matter base material the porous matter film of porous matter resin layer.
Water: 30.0 weight %
DMAc:70.0 weight %.
After this porous matter film peeled off from sheet glass, impregnated in the hot water of 80 ℃ of temperature 3 times, wash out DMAc, obtained separatory membrane (porous membrane).The scope of observing porous matter resin layer surface 9.2 μ m * 10.4 μ m with 10,000 times multiplying power with scanning electron microscope, diameter mean value that can observed all pores is 0.1 μ m.Then, the pure water transmission coefficient of above-mentioned separatory membrane is estimated, the result is 50 * 10 -9m 3/ m 2SPa.The mensuration of the permeable amount of the pure water purified water of 25 ℃ of temperature that obtain by reverse osmosis membrane is carried out with the drop height of 1m.In addition, the standard deviation of fine pore is 0.035 μ m, and the film surfaceness is 0.06 μ m.
(embodiment 1)
Use the SU014 strain of reference example 1 preparation, carry out cultured continuously, make L-lactic acid by bactogen shown in Figure 2.And, use raw material sugar culture-medium (the excellent asccharin of 60g/L (system ソ one Co., Ltd.'s system), 1.5g/L ammonium sulfate) as substratum.This raw material sugar culture-medium re-uses after 121 ℃ temperature high pressure (2 normal atmosphere) steam sterilizing is handled 15 minutes.The separating film element material uses the moulding product of stainless steel and polysulfone resin, the flat film of porousness that separatory membrane uses reference example 2 to make.Use diaphragm pump " APLS-20 " (タ Network ミ Na society) as the pump 5 in the liquid pushing tube 17, use peristaltic pump to take out the pump 4 that sees through liquid from the membrane sepn groove as being used for.And the operating condition among the embodiment is as follows.
Culture tank capacity: 20 (L)
Use separatory membrane: PVDF filtering membrane (making in the reference example 2)
Membrane sepn tankage: 5 (L)
Membrane sepn element effective filtration area: 4000cm 2
Temperature regulation: 32 (℃)
Culture tank air flow: air 1 (L/ minute)
Culture tank stirring velocity: 100 (rpm) pH regulator: be adjusted to pH5 with 8N calcium hydroxide
Substratum feed speed: with the water level sensor 12 variable controls in the culture tank
Sterilization: membrane sepn groove, culture tank and use substratum all adopt 121 ℃, 0.2MPa, 20 minutes steam under pressure sterilization
The flow of pump 4: 3L/ hour
The maximum inner diameter of liquid pushing tube 15,17: 50mm
The output of pump 5: 5L/ minute
The linear flow speed of liquid pushing tube 15,17: 4.2cm/ second
Filter flow: 0.180m/ days
The rate of recovery: non-control (below 1%).
So,, in test tube, use one night of raw material sugar culture-medium shaking culture SU014 strain (preceding cultivation) of 5ml as preceding cultivation.The nutrient solution that inoculation obtains in the fresh raw material sugar culture-medium of 100ml holds slope mouth flask, 30 ℃ of shaking culture 24 hours (preceding cultivation) with 500ml.The nutrient solution that obtains is seeded in the fresh raw material sugar culture-medium of 1000ml, holds slope mouth flask, 30 ℃ of shaking culture 24 hours (preceding cultivation) with 3000ml.
This preceding nutrient solution is seeded in the lactic fermentation substratum of the total 20L in culture tank 1 and the membrane sepn groove, in incidental stirrer stir culture groove, carry out adjusting, temperature regulation, the pH regulator of air flow, after carrying out cultivation in 50 hours, make pump 4 runnings, take out the liquid that sees through that contains L-lactic acid.At this moment, measure the pressure of the nutrient solution that flows into to membrane sepn groove 2 once a day, regulate the flow rate control device 25 (butterfly valve) that is arranged in the bypass pipe 26, make gauge pressure reach 0.1MPa.
Carry out cultivating in 250 hours, measure yeast turbidity in the culture tank, as the lactic acid concn, the sugared concentration that see through the production material in the liquid, calculate lactic acid to sugared yield.It the results are shown in Figure 10, table 1.And lactic acid concn is measured with the method shown in the reference example 1.The yeast turbidity is by measuring at the extinction of 600nm with photometer.In addition, so-called lactic acid to sugared yield, be the lactic acid weight produced ratio with respect to the sugar weight that consumes, calculate with following (formula 7).
[several 7]
Figure BDA0000052877920000441
The mensuration of sugar concentration is measured by the HPLC method under the following conditions.
Post: Luna NH2 250x4.6mm (Phenomenex corporate system)
Mobile phase: water: acetonitrile=25: 75
Flow velocity: 0.6ml/ minute
Detection method: RI (differential refractometer)
Reply: 4
Polarity :+
Temperature: 30 ℃.
(comparative example 1)
Except using bactogen shown in Figure 9, all the other modes the same with embodiment 1 are carried out cultured continuously, measure the yeast turbidity, as the lactic acid concn of producing material.And except that device shown in Figure 9 was not provided with bypass pipe 26, flow rate control device 25 and membrane sepn groove threshold switch 27,28, all the other were the same with the formation of device shown in Figure 2.
The results are shown in Figure 11, table 1.And, to the Figure 12 that the results are shown in of the piezometry of the nutrient solution supplied with to the membrane sepn groove in the cultured continuously.
In the comparative example 1, owing to do not control the pressure of the nutrient solution of supplying with to the membrane sepn groove, therefore as shown in figure 12, pressure changes in cultured continuously, changes to more than the 1MPa in the time of 250 hours from cultivating beginning.And the lactic acid concn of yeast turbidity and production all is lower than embodiment 1, lactic acid be 63% after 250 hours cultured continuously to sugared yield.
According to above content, can confirm to have produced zymic high-density culture, the raising of lactic acid (chemical) concentration and lactic acid sugared yield is improved the effect that these can't be envisioned owing to regulate the inflow of nutrient solutions to the membrane sepn groove with bypass pipe 26 and the flow rate control device 25 that is arranged on wherein.
(embodiment 2)
Use and to have finished bactogen and the nutrient solution cultivated among the embodiment 1, with the linear flow speed in the circulation tube reach 0.5,1.5,2.5cm/ second, carry out 2 hours liquor chargings respectively, measure and be deposited in the pipe arrangement amount of cumulative thalline.It the results are shown in Fig. 6.We can say like this, reach 2.5cm/ more than second, can not be deposited in mode in the pipe arrangement nutrient solution that circulates with thalline by making the nutrient solution linear velocity in the circulation tube.
(embodiment 3)
Except the output of pump 5 was changed into 10L/ minute, all the other carried out cultured continuously in mode similarly to Example 1.Mensuration carry out after 100 hours the cultivation and carry out 250 hours cultivation after culture tank in the yeast turbidity, as the lactic acid concn, the sugared concentration that see through the production material in the liquid, calculate again lactic acid to sugared yield.The results are shown in table 1.
Embodiment 3 will reduce sugared yield than the lactic acid concn of embodiment 1, lactic acid.We think that this is because by making circular flow (pump 5) increase, probably variation has taken place the liquid mixing state of culture tank.
(comparative example 2)
Except using the bactogen shown in Fig. 9, all the other are all to carry out cultured continuously with embodiment 3 same way as.
Because in comparative example 2, do not control the pressure of the nutrient solution of supplying with to the membrane sepn groove, therefore membrane sepn groove internal pressure increases in the cultured continuously, cultivating beginning reached more than the 1MPa in the time of 70 hours, if remain in operation again, nutrient solution just begins to leak from the membrane sepn groove, has formed the state that can't implement cultured continuously.
By embodiment 3 and comparative example 2, known bypass conduit-free 26, just can't implement cultured continuously, regulate the inflow of nutrient solution, we can say to have the effect that stably to implement cultured continuously to the membrane sepn groove by the flow rate control device 25 that is arranged in the bypass pipe 26.
(embodiment 4)
Use bactogen shown in Figure 7, except pump 5 is output as 5L/ minute, beyond pump 16 was output as 10L/ minute, all the other modes the same with embodiment 3 were carried out cultured continuously.
Mensuration carried out cultivating the back in 100 hours and cultivated in 250 hours after culture tank in the yeast turbidity, as lactic acid concn, sugared concentration through the production material in the liquid, calculate again lactic acid to sugared yield.The results are shown in table 1.
Its result, opposite with embodiment 1, even by pump 16, increase circular flow similarly to Example 3, owing to return the flow of the liquid of culture tank by pump 16 control, lactic acid concn, yeast turbidity and lactic acid also can reach the equal achievement of embodiment 1 before changing with circular flow to sugared yield.
(comparative example 3)
Except using bactogen shown in Figure 13, all the other modes identical with embodiment 4 are carried out cultured continuously.And except device shown in Figure 13 was not provided with bypass pipe 26, flow rate control device 25 and membrane sepn groove valve 27,28, all the other structures were identical with device shown in Figure 7.
Owing to do not control the pressure of the nutrient solution of supplying with to the membrane sepn groove in the comparative example 3, therefore pressure increases in the cultured continuously, reaches more than the 1MPa when cultivating 70 hours that begin, if remain in operation again, nutrient solution just begins to leak from the membrane sepn groove, has formed the state that can't implement cultured continuously.
(embodiment 5)
Except using bactogen shown in Figure 14, all the other similarly to Example 3 mode carry out cultured continuously.And the device shown in Figure 14 position opening in the nutrient solution that liquid pushing tube 15 is stored in impregnated in culture tank 1, all the other structures are identical with device shown in Figure 2.
Mensuration carried out cultivating the back in 100 hours and cultivated in 250 hours after culture tank in the yeast turbidity, as lactic acid concn, sugared concentration through the production material in the liquid, calculate again lactic acid to sugared yield.The results are shown in table 1.
Its result, opposite with embodiment 1, even by pump 5, increase circular flow similarly to Example 3, owing to be arranged on the position that impregnated in the nutrient solution by the position that liquid is turned back to culture tank, lactic acid concn, yeast turbidity and lactic acid also can reach the equal achievement of embodiment 1 before changing with circular flow to sugared yield.
(comparative example 4)
Except using bactogen shown in Figure 15, all the other modes identical with embodiment 5 are carried out cultured continuously.And except device shown in Figure 15 was not provided with bypass pipe 26, flow rate control device 25 and membrane sepn groove switch- valve 27,28, all the other structures were identical with device shown in Figure 14.
Owing to do not control the pressure of the nutrient solution of supplying with to the membrane sepn groove in the comparative example 4, therefore pressure increases in the cultured continuously, reaches more than the 1MPa when cultivating 70 hours that begin, if remain in operation again, nutrient solution just begins to leak from the membrane sepn groove, has formed the state that can't implement cultured continuously.
[table 1]
Figure BDA0000052877920000481
(embodiment 6)
Use bactogen shown in Figure 16, value calculate recovery rate according under meter 30, seeing through liquid taking-up flow by pump 4 adjustings makes the rate of recovery reach 1.5%, meanwhile carry out cultured continuously, and after 250 hours, continue outside the cultured continuously all the other and the embodiment 1 the same cultured continuously of carrying out.And device shown in Figure 16 is except being provided with under meter 30, and all the other are identical with device shown in Figure 2.Simultaneously, through the time measure the intermembranous differential pressure act on separatory membrane 3, estimate the off-period of the film that causes from rising suddenly of intermembranous differential pressure.
The passing of the intermembranous differential pressure of measuring has been shown among Figure 17.Begin through 1000 hours from running, intermembranous differential pressure is stable, and the rate of recovery running with 1.5% can realize making L-lactic acid by cultured continuously long-term and stably.Yeast turbidity, conduct see through the table 2 that the results are shown in to sugared yield of the lactic acid concn of the production material in the liquid, sugared concentration, lactic acid in the culture tank of measuring when in addition, cultured continuously is finished and calculating.
(embodiment 7)
Except the rate of recovery is 3.0%, all the other similarly to Example 6 mode carry out cultured continuously.
The passing of the intermembranous differential pressure of measuring has been shown among Figure 17.Begin through 800 hours from running, intermembranous differential pressure is stable, even turn round with 3.0% the rate of recovery, also can realize making L-lactic acid by cultured continuously long-term and stably.Yeast turbidity, conduct see through the table 2 that the results are shown in to sugared yield of the lactic acid concn of the production material in the liquid, sugared concentration, lactic acid in the culture tank of measuring when in addition, cultured continuously is finished and calculating.
(embodiment 8)
Except the rate of recovery becomes 9.9%, all the other similarly to Example 6 mode carry out cultured continuously.
The passing of the intermembranous differential pressure of measuring has been shown among Figure 17.Begin through 550 hours from running, intermembranous differential pressure is stable, even in the rate of recovery 9.9%, also can realize stably making L-lactic acid by cultured continuously.Yeast turbidity, conduct see through the table 2 that the results are shown in to sugared yield of the lactic acid concn of the production material in the liquid, sugared concentration, lactic acid in the culture tank of measuring when in addition, cultured continuously is finished and calculating.
(embodiment 9)
Except the rate of recovery becomes 12.0%, all the other similarly to Example 6 mode carry out cultured continuously.
The passing of the intermembranous differential pressure of measuring has been shown among Figure 17.In the time of 100 hours, intermembranous differential pressure sharply rises from the running beginning, and the film pore is closed.Yeast turbidity, conduct see through the table 2 that the results are shown in to sugared yield of the lactic acid concn of the production material in the liquid, sugared concentration, lactic acid in the culture tank of measuring when in addition, cultured continuously is finished and calculating.Carrying out 100 hours lactic acid concns in the culture tank after the cultured continuously is 45g/L.And it is 100 that the yeast turbidity is increased to OD600, lactic acid be 80% to sugared yield.
Yet, filtered the change difficulty, surpass 100 hours, be difficult to continue to make L-lactic acid by cultured continuously.
More than, according to the result of embodiment 6～9, can confirm, by carrying out the cultured continuously running, can carry out this unforeseen unusual effect of the cultured continuously running of long-time (more than 500 hours) in the mode that the rate of recovery is adjusted to below 10%.
[table 2]
Implementation condition Embodiment 6 Embodiment 7 Embodiment 8 Embodiment 9
Chemical Lactic acid Lactic acid Lactic acid Lactic acid
Microorganism SU014 SU014 SU014 SU014
Device Figure 16 Figure 16 Figure 16 Figure 16
Pump 4 Change Change Change Change
Pump
5 5L/min 5L/min 5L/min 5L/min
Pump 16 - - - -
Filter flow Change Change Change Change
The rate of recovery 1.50％ 3.00％ 9.90％ 12.00％
Incubation time 1000h 800h 550h 100h
Compound concentration 45g/L 45g/L 45g/L 45g/L
Microorganism concn 320 270 250 100
To sugared yield 80％ 80％ 80％ 80％
(embodiment 10)
Use the spy to open the described Corynebacterium glutamicum TR-CAD1 of 2004-222569 communique strain, carry out cultured continuously, make cadaverine by bactogen shown in Figure 2.And, use the cadaverine of forming shown in the table 3 to produce substratum as substratum.Temperature high pressure (2 normal atmosphere) the steam sterilizing processing that this cadaverine production is cultivated based on 121 ℃ re-used after 15 minutes.The separating film element material uses the moulding product of stainless steel and polysulfone resin, the flat film of porousness that separatory membrane uses reference example 2 to make.Use diaphragm pump " APLS-20 " (タ Network ミ Na society) as the pump 5 in the liquid pushing tube 17, use peristaltic pump to take out the pump 4 that sees through liquid from the membrane sepn groove as being used for.
[table 3]
Figure BDA0000052877920000511
And the condition among the embodiment is as follows.
Culture tank capacity: 20 (L)
Use separatory membrane: PVDF filtering membrane (making in the reference example 2)
Membrane sepn tankage: 5 (L)
Membrane sepn element effective filtration area: 4000cm 2
Temperature regulation: 30 (℃)
Culture tank air flow: air 3 (L/ minute)
Culture tank stirring velocity: 100 (rpm) pH regulator: be adjusted to pH7.0 with 3M HCl and 3M ammoniacal liquor
Substratum feed speed: with the variable control of liquid level water level inductor block in the culture apparatus
Sterilization: membrane sepn groove, culture tank and use substratum all adopt 121 ℃, 0.2MPa, 20 minutes steam under pressure sterilization
The flow of pump 4: 3L/ hour
The maximum inner diameter of liquid pushing tube 15,17: 50mm
The output of pump 5: 5L/ minute
The linear flow speed of liquid pushing tube 15,17: 4.2cm/ second
Filter flow: 0.180m/ days
The rate of recovery: non-control (below 1%).
So,, in test tube, produce substratum shaking culture TR-CAD1 strain one evening (preceding cultivation) with the cadaverine that has added 5ml kantlex (25 μ g/ml) as preceding cultivation.In the fresh interpolation of 50ml the cadaverine of kantlex (25 μ g/ml) produce the nutrient solution that inoculation of medium obtains, hold slope mouth flask with 500ml, 30 ℃ temperature,, cultivate 24 hours (preceding cultivation) with amplitude 30cm, under the condition of 180rpm.The nutrient solution that obtains is seeded in the fresh cadaverine of 1000ml produces in the substratum, hold slope mouth flask, 30 ℃ of shaking culture 24 hours (preceding cultivation) with 3000ml.The cadaverine that this preceding nutrient solution is seeded in the total 20L in culture tank 1 and the membrane sepn groove 2 is produced in the substratum, in incidental stirrer stir culture groove, carry out adjusting, temperature regulation, the pH regulator of air flow, after carrying out cultivation in 50 hours, make pump 4 runnings, take out the liquid that sees through that contains cadaverine.
At this moment, measure the pressure of the nutrient solution that flows into to membrane sepn groove 2 once a day, regulate the flow rate control device 25 (butterfly valve) that is arranged in the bypass pipe 26, make gauge pressure reach 0.1MPa.
Carry out cultivating in 250 hours, measure yeast turbidity in the culture tank, as the cadaverine concentration, the sugared concentration that see through the production material in the liquid, calculate again cadaverine to sugared yield.It the results are shown in Figure 18, the table 4.Cadaverine concentration is 3.5g/L.In addition, excellent bacillus turbidity is by measuring at the extinction of 600nm with photometer.Cadaverine be 3% to sugared yield.
And cadaverine concentration is measured in order to following method.
[analytical procedure of the cadaverine concentration of being undertaken by HPLC]
Use post: CAPCELL PAK C18 (Shiseido)
Mobile phase: 0.1% (w/w) phosphate aqueous solution: acetonitrile=4.5: 5.5
Detect: UV360nm
The sample pre-treatment: in 25 μ l analytical samples,, add and mix 25 μ l 1 as interior mark, 4-diaminobutane (0.03M), the sodium bicarbonate of 150 μ l (0.075M) and 2, the ethanolic soln of 4-dinitrofluorobenzene (0.2M) was in 37 ℃ temperature insulations 1 hour.
After above-mentioned reaction soln 50 μ l were dissolved in the acetonitrile of 1ml, with 10,000rpm was after centrifugal 5 minutes, its supernatant of 10 μ l carried out HPLC analyze.
(comparative example 5)
Except using device shown in Figure 9, all the other modes the same with embodiment 10 are carried out cultured continuously, measure excellent bacillus turbidity, as the cadaverine concentration of producing material.And except that device shown in Figure 9 was not provided with bypass pipe 26, flow rate control device 25 and membrane sepn groove threshold switch 27,28, all the other were the same with the formation of device shown in Figure 2.
The results are shown in Figure 19, table 4.And, to the Figure 20 that the results are shown in of the piezometry of the nutrient solution supplied with to the membrane sepn groove in the cultured continuously.
[table 4]
Implementation condition Embodiment 10 Comparative example 5
Chemical Cadaverine Cadaverine
Microorganism TR-CAD1 TR-CAD1
Device Fig. 2 Fig. 9
Pump 4 3L/hr 3L/hr
Pump
5 5L/min 5L/min
Pump 16 - -
Filter flow 0.180m/ my god 0.180m/ my god
The rate of recovery Below 1% 1％
Incubation time 250h 250h
Compound concentration 3.5g/L 1.2g/L
Microorganism concn
250 100
To sugared yield 3％ 1％
Owing to do not control the pressure of the nutrient solution of supplying with to the membrane sepn groove in the comparative example 5, therefore as shown in figure 20, pressure change in the cultured continuously reaches more than the 1MPa when 225 hours of cultivating after beginning.And excellent bacillus turbidity, cadaverine concentration all are lower than embodiment 10.And, cadaverine be 1.0% to sugared yield.
Can confirm by above, owing to regulate the inflow of nutrient solutions to the membrane sepn groove with bypass pipe 26 and the flow rate control device 25 that is arranged on wherein, the high-density culture of excellent bacillus, the raising of cadaverine (chemical) concentration and cadaverine sugared yield is improved the effect that these can't be envisioned.
(embodiment 11)
Use the spy to open the described Corynebacterium glutamicum Δ of 2008-212138 communique-HOM strain, carry out cultured continuously, make L-Methionin by bactogen shown in Figure 2.And, use the L-Methionin of forming shown in the table 5 to produce substratum as substratum.Temperature high pressure (2 normal atmosphere) the steam sterilizing processing that this L-Methionin production is cultivated based on 121 ℃ re-used after 15 minutes.The separating film element material uses the moulding product of stainless steel and polysulfone resin, the flat film of porousness that separatory membrane uses reference example 2 to make.Use diaphragm pump " APLS-20 " (タ Network ミ Na society) as the pump 5 in the liquid pushing tube 17, use peristaltic pump to take out the pump 4 that sees through liquid from the membrane sepn groove as being used for.
[table 5]
Figure BDA0000052877920000541
And the condition among the embodiment is as follows.
Culture tank capacity: 20 (L)
Use separatory membrane: PVDF filtering membrane (making in the reference example 2)
Membrane separation unit liquid measure: 5 (L)
Membrane sepn element effective filtration area: 4000cm 2
Temperature regulation: 30 (℃)
Culture tank air flow: air 5 (L/ minute)
Culture tank stirring velocity: 300 (rpm) pH regulator: be adjusted to pH7.3 with 3M HCl and 3M ammoniacal liquor
Substratum feed speed: with the variable control of liquid level water level inductor block in the culture apparatus
Sterilization: membrane sepn groove, culture tank and use substratum all adopt 121 ℃, 0.2MPa, 20 minutes steam under pressure sterilization
The flow of pump 4: 3L/ hour
The maximum inner diameter of liquid pushing tube 15,17: 50mm
The output of pump 5: 5L/ minute
The linear flow speed of liquid pushing tube 15,17: 4.2cm/ second
Filter flow: 0.180m/ days
The rate of recovery: non-control (below 1%).
So,, in test tube, use BY substratum (0.5% yeast extract, 0.7% meat extract, 1% peptone, 0.3% sodium-chlor) shaking culture Δ-HOM strain one evening (preceding cultivation) of 5ml as preceding cultivation.The L-Methionin that the nutrient solution that obtains is seeded in 50ml is produced in the substratum, holds slope mouth flask with 500ml, 30 ℃ temperature, with amplitude 30cm, under the condition of 180rpm, cultivates 24 hours (preceding cultivation).The nutrient solution that obtains is seeded in the fresh L-Methionin of 1000ml produces in the substratum, hold slope mouth flask, 30 ℃ of shaking culture 24 hours (preceding cultivation) with 3000ml.The L-Methionin that this preceding nutrient solution is seeded in the 20L in culture tank 1 and the membrane sepn groove is produced in the substratum, in incidental stirrer stir culture groove, carry out adjusting, temperature regulation, the pH regulator of air flow, after carrying out cultivation in 50 hours, make pump 4 runnings, take out the liquid that sees through that contains L-Methionin.
At this moment, measure the pressure of the nutrient solution that flows into to membrane sepn groove 2 once a day, regulate the flow rate control device 25 (butterfly valve) that is arranged in the bypass pipe 26, make gauge pressure reach 0.1MPa.
Carry out cultivating in 250 hours, measure excellent bacillus turbidity in the culture tank, as the L-lysine concentration, the sugared concentration that see through the production material in the liquid, calculate again the L-lysine concentration to sugared yield.It the results are shown in Figure 21, table 6.The L-lysine concentration is 6.0g/L.In addition, excellent bacillus turbidity is by measuring at the extinction of 600nm with photometer.L-Methionin be 5.5% to sugared yield.In addition, the L-lysine concentration is measured by the method identical with the assay method of cadaverine concentration.
(comparative example 6)
Except using device shown in Figure 9, all the other modes are similarly to Example 11 carried out cultured continuously, measure the L-lysine concentration of excellent bacillus turbidity, conduct production material.And except that device shown in Figure 9 was not provided with bypass pipe 26, flow rate control device 25 and membrane sepn groove threshold switch 27,28, all the other were the same with the formation of device shown in Figure 2.
The results are shown in Figure 22, table 6.And, to the Figure 23 that the results are shown in of the piezometry of the nutrient solution supplied with to the membrane sepn groove in the cultured continuously.
[table 6]
Implementation condition Embodiment 11 Comparative example 6
Chemical L-Methionin L-Methionin
Microorganism Δ-HOM Δ-HOM
Device Fig. 2 Fig. 9
Pump 4 3L/hr 3L/hr
Pump
5 5L/ minute 5L/ minute
Pump 16 - -
Filter flow 0.180m/ my god 0.180m/ my god
The rate of recovery Below 1% 1％
Incubation time 250h 250h
Compound concentration 6.0g/L 1.2g/L
Microorganism concn 250 100
To sugared yield 5.5％ 1.1％
Owing to do not control the pressure of the nutrient solution of supplying with to the membrane sepn groove in the comparative example 6, therefore as shown in figure 23, pressure change in the cultured continuously reaches more than the 1MPa when 225 hours of cultivating after beginning.And excellent bacillus turbidity, L-lysine concentration all are lower than embodiment 11.And, L-Methionin be 1.1% to sugared yield.
Can confirm by above, owing to regulate the inflow of nutrient solutions to the membrane sepn groove with bypass pipe 26 and the flow rate control device 25 that is arranged on wherein, produce excellent bacillus high-density culture, the raising of L-Methionin (chemical) concentration and L-Methionin sugared yield is improved the effect that these can't be envisioned.
Industrial applicibility
The present invention can be used for alcohol, organic acid, amino acid, nucleic acid, enzyme, antibiotic, recombinant protein etc. are cultivated the various chemicals that can obtain by microorganism production suitably.
Symbol description
1. culture tank
2,2 '. the film separating tank
3,3 '. diffusion barrier
4. filter pump
5. pump
6. culture medium supply pump
7. shaft
8. gas supply pipe
9.pH sensor
10. nertralizer pump
11. thermoregulator
12. water level sensor
13. atmospheric pressure opening portion
14A. point
14B. branch point
15. liquid pushing tube (not filtering nutrient solution returns to culture tank)
16. pump
17. liquid pushing tube
18. support plate
19. flow channel material
20. diffusion barrier
21. recess
22. collector tube
23. upper resin sealing ply
24. the resin-sealed layer in bottom
25. flow rate control device
26. bypass pipe
27,27 '. membrane sepn groove open and close valve (substratum supply side)
28,28 '. membrane sepn groove open and close valve (substratum discharge side)
29. pressure warning unit
30. under meter
Figure IDA0000052878010000011
Figure IDA0000052878010000021
Figure IDA0000052878010000031

Claims (14)

1. make a part by the nutrient solution of culture tank liquor charging, nutrient solution inflow side pressure according to the membrane sepn groove, walk around the manufacture method of the chemical of membrane sepn groove, this method is culturing micro-organisms or a culturing cell in culture tank, carry nutrient solution by culture tank to the membrane sepn groove, filter with separatory membrane again, from see through liquid, reclaim fermentative production thing, the nutrient solution that do not filter of filtered is refluxed in the mode that the nutrient solution with membrane sepn groove upstream side converges as chemical.
2. the manufacture method of chemical according to claim 1, its gauge pressure with the nutrient solution inflow side of membrane sepn groove be below the 1MPa like that, the flow of the nutrient solution of membrane sepn groove is walked around in control.
3. the manufacture method of chemical according to claim 1 and 2, its make a part of not filtering nutrient solution with culture tank in the mode converged of nutrient solution reflux, make simultaneously the rest part that do not filter nutrient solution with and culture tank to the mode that the nutrient solution between the membrane sepn groove converges reflux.
4. the manufacture method of chemical according to claim 3, its control independently of each other with and culture tank to the nutrient solution between the membrane sepn groove converge the flow that does not filter nutrient solution that mode refluxes and with culture tank in nutrient solution converge the flow that does not filter nutrient solution that mode refluxes.
5. the manufacture method of claim 3 or 4 described chemical, with and culture tank in nutrient solution converge the flow that does not filter nutrient solution that mode refluxes with respect to and culture tank to the nutrient solution between the membrane sepn groove converge the ratio of the flow that does not filter nutrient solution that mode refluxes below 1.
6. according to the manufacture method of each described chemical of claim 1～5, the linear flow speed of the linear flow speed of the nutrient solution of being carried to the membrane sepn groove by culture tank, the linear flow speed of not filtering nutrient solution that refluxes in the mode of being converged to the nutrient solution of this membrane sepn groove upstream side by the membrane sepn groove and the nutrient solution of walking around the membrane sepn groove is all at 2.5cm/ more than second.
7. according to the manufacture method of each described chemical of claim 1～6, regulate to flow into the liquid measure that sees through of the cultivation liquid measure of membrane sepn groove and/or self-separation film, so that the liquid measure that sees through of self-separation film is below 10.0% with respect to the rate of recovery of the cultivation liquid measure that flows into the membrane sepn groove.
8. according to the manufacture method of each described chemical of claim 1～7, in the culture tank in nutrient solution volume and the membrane sepn groove ratio of nutrient solution volume be more than 4 below 100.
9. the bypass pipe that possesses the membrane sepn groove, the proofing unit of the nutrient solution inflow side pressure of membrane sepn groove, bactogen with the flow rate control device that is arranged on bypass pipe, this bactogen possesses the culture tank that is used for culturing micro-organisms or culturing cell, the membrane sepn groove that possesses the separatory membrane that is used for retrieving the fermentative production thing produced from the nutrient solution of culture tank, connect culture tank and membrane sepn groove and when nutrient solution is transported to the aforementioned films separator tank, make without filtering circulation tube that mode that nutrient solution converges with the nutrient solution with membrane sepn groove upstream side refluxes and the nutrient solution liquid feeding device that is arranged on circulation tube of not filtering of separatory membrane.
10. bactogen according to claim 9, described flow rate control device are to be turned round by result's interlock of proofing unit.
11. according to claim 9 or 10 described bactogens, it possesses the linear flow speed detector of circulation tube, flow rate control device and/or aforementioned nutrient solution liquid feeding device are that the result according to this linear flow speed detector turns round.
12. according to each described bactogen of claim 9～11, described membrane sepn groove be installed in have the liquid feeding device different with aforementioned nutrient solution liquid feeding device and with culture tank independently in the circulation loop.
13. according to each described bactogen of claim 9～12, the position opening in the nutrient solution that circulation tube retains in impregnated in culture tank.
14. according to each described bactogen of claim 9～13, the long-pending ratio of culture tank volume and membrane sepn cell body is more than 4 below 100.
CN2009801386054A 2008-09-30 2009-09-16 Method for producing chemical and continuous culture system Pending CN102171349A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610340983.2A CN106011187A (en) 2008-09-30 2009-09-16 Method for producing chemical product and continuous fermentation apparatus

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
JP2008252724 2008-09-30
JP252723/2008 2008-09-30
JP252724/2008 2008-09-30
JP2008252723 2008-09-30
PCT/JP2009/066119 WO2010038613A1 (en) 2008-09-30 2009-09-16 Method for producing chemical and continuous culture system

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN201610340983.2A Division CN106011187A (en) 2008-09-30 2009-09-16 Method for producing chemical product and continuous fermentation apparatus

Publications (1)

Publication Number Publication Date
CN102171349A true CN102171349A (en) 2011-08-31

Family

ID=42073382

Family Applications (2)

Application Number Title Priority Date Filing Date
CN2009801386054A Pending CN102171349A (en) 2008-09-30 2009-09-16 Method for producing chemical and continuous culture system
CN201610340983.2A Pending CN106011187A (en) 2008-09-30 2009-09-16 Method for producing chemical product and continuous fermentation apparatus

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201610340983.2A Pending CN106011187A (en) 2008-09-30 2009-09-16 Method for producing chemical product and continuous fermentation apparatus

Country Status (11)

Country Link
US (2) US20110177551A1 (en)
EP (1) EP2330209B1 (en)
JP (1) JP5881135B2 (en)
KR (1) KR20110060888A (en)
CN (2) CN102171349A (en)
AU (1) AU2009299152B2 (en)
BR (1) BRPI0913137B1 (en)
CA (1) CA2738981C (en)
PL (1) PL2330209T3 (en)
RU (1) RU2513694C2 (en)
WO (1) WO2010038613A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110832081A (en) * 2017-06-30 2020-02-21 东丽株式会社 Method and apparatus for producing chemical by continuous fermentation

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8999681B2 (en) * 2010-12-08 2015-04-07 Toray Industries, Inc. Method for producing cadaverine
JP5742327B2 (en) * 2011-03-15 2015-07-01 東レ株式会社 Continuous fermentation weight control device, continuous fermentation system, continuous fermentation weight control method, and continuous fermentation weight control program
KR101286158B1 (en) * 2011-08-24 2013-07-15 씨제이제일제당 (주) The seperation and purification method of 1,4-diaminobutane from a fermented solution
HUE052865T2 (en) * 2011-09-01 2021-05-28 Gicon Grossmann Ingenieur Consult Gmbh Method and device for feeding gases or gas mixtures into a liquid, suspension, or emulsion in a photobioreactor in a specific manner
US10378029B2 (en) * 2012-01-13 2019-08-13 Toray Industries, Inc. Method of producing chemical substance
US9644221B2 (en) 2012-03-30 2017-05-09 Toray Industries, Inc. Method of producing chemical by continuous fermentation and continuous fermentation apparatus
EP2708598A1 (en) * 2012-09-14 2014-03-19 Basf Se Serinol production in glycerol catabolism deficient escherichia coli strains
JP6467735B2 (en) * 2014-07-18 2019-02-13 佐竹化学機械工業株式会社 Reciprocating agitation culture device with medium extraction function
KR20160097691A (en) 2015-02-09 2016-08-18 씨제이제일제당 (주) The novel Lysine Decarboxylase and Process for producing cadeverine using the same
WO2016203586A1 (en) 2015-06-17 2016-12-22 日立化成株式会社 Polishing agent, storage solution for polishing agent and polishing method
CN107849513A (en) * 2015-06-23 2018-03-27 明尼苏达-达科塔农民合作社 The method of yeast autolysis through saponin(e enhancing
JP6142042B1 (en) 2016-03-18 2017-06-07 株式会社村田製作所 Filtration filter for nucleated cells and filtration method using the same
JP6682172B2 (en) 2017-03-03 2020-04-15 富士フイルム株式会社 Cell culture device and cell culture method
JP6249124B1 (en) 2017-04-26 2017-12-20 株式会社村田製作所 Filtration filter for nucleated cells and filtration method using the same
WO2019009044A1 (en) * 2017-07-03 2019-01-10 株式会社村田製作所 Concentrating device
US11584907B2 (en) 2017-09-13 2023-02-21 National Institute Of Advanced Industrial Science And Technology Cell culture apparatus and cell culture method
JP6815307B2 (en) * 2017-11-09 2021-01-20 株式会社村田製作所 Filtration filter for nucleated cells and filtration method using it
KR101976955B1 (en) * 2017-11-17 2019-05-09 영남대학교 산학협력단 Culture system for bacteriophage
TWI724324B (en) * 2017-12-21 2021-04-11 鼎唐能源科技股份有限公司 Method for producing butyric acid and/or its salts
JP7087704B2 (en) * 2018-06-13 2022-06-21 株式会社Ihi Cell culture system and cell culture method
CN110117527A (en) * 2019-05-15 2019-08-13 刘宝全 A kind of reinforcing discharge method of stem cell metabolic waste
JP2021045100A (en) * 2019-09-20 2021-03-25 株式会社日立製作所 Cell separation device and cell separation method
GB2593223A (en) * 2020-03-20 2021-09-22 Dairy Crest Ltd Reactor
CN112063662B (en) * 2020-09-22 2022-07-05 宁波海硕药业科技有限公司 Production method and equipment of L-leucine
CN114045225B (en) * 2021-11-22 2023-03-10 广西科技师范学院 Candida glabrata SLLSM3 and application thereof
CN114134036B (en) * 2021-12-15 2023-03-10 徐州生物工程职业技术学院 Production equipment and process of small molecule peptide medicine
US20230358651A1 (en) * 2022-05-09 2023-11-09 Robert G. Saunders Device Used for Real Time Field Sampling for DNA Extraction
CN115536445A (en) * 2022-09-07 2022-12-30 江苏太古合一环境管理有限公司 Method for preparing liquid organic base fertilizer from kitchen waste, liquid organic base fertilizer and liquid organic fertilizer
WO2024106442A1 (en) * 2022-11-15 2024-05-23 旭化成メディカル株式会社 Evaluation method of filtration condition and production method of cell product

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1284077A (en) * 1968-10-18 1972-08-02 British Petroleum Co Process and apparatus for the cultivation of micro-organisms
SU440021A1 (en) * 1970-10-28 1975-03-15 Предприятие П/Я В-2262 Apparatus for receiving and processing chemical products
JPS557095A (en) 1979-06-04 1980-01-18 Tsukuba Daigaku Production of itaconic acid
DE3005605A1 (en) * 1980-02-15 1981-10-01 Sartorius GmbH, 3400 Göttingen Antigen extn. from cell suspension - by membrane and ultrafiltration stages
DE3477399D1 (en) * 1983-07-27 1989-04-27 Mueller Drm Ag Method for the separation of yeast from fermentation broths and application of the method
DE3783081T2 (en) * 1986-06-11 1993-04-15 Michigan Biotech Inst METHOD FOR PRODUCING AMBER ACID BY ANAEROBIC FERMENTATION.
US6022742A (en) * 1986-11-26 2000-02-08 Kopf; Henry B. Culture device and method
SU1719433A1 (en) 1988-04-26 1992-03-15 Научно-Исследовательский Технологический Институт Аминокислот Method of l-alanine preparation
JPH0646937B2 (en) * 1988-10-17 1994-06-22 雪印乳業株式会社 Concentrator for culture fluid
JPH0632626B2 (en) 1989-02-20 1994-05-02 東レ株式会社 Fermentation method for producing L-threonine
JP3243891B2 (en) 1993-06-14 2002-01-07 東レ株式会社 Purification method of pyruvate
AT402635B (en) * 1993-10-21 1997-07-25 A Abfall Service Holding Ges M Plant and process for the microbiological treatment of liquid media
US5770435A (en) * 1995-11-02 1998-06-23 University Of Chicago Mutant E. coli strain with increased succinic acid production
US6149824A (en) * 1996-08-22 2000-11-21 Water Refining, Inc. Continuous filtration system using single pump, venturi, and flow control valve
RU2123380C1 (en) * 1996-10-22 1998-12-20 Ангарский электролизный химический комбинат Reactor for chemical products
JP3812019B2 (en) * 1996-11-21 2006-08-23 味の素株式会社 Process for producing L-glutamic acid by continuous fermentation
US8026096B1 (en) * 1998-10-08 2011-09-27 Protein Sciences Corporation In vivo active erythropoietin produced in insect cells
JP2004222569A (en) 2003-01-22 2004-08-12 Toray Ind Inc Corynebacterium, and cadaverine or salt thereof and method for producing the same
US20040259240A1 (en) * 2003-06-17 2004-12-23 Fadden Stephen J. Method and apparatus for filtration of bioreactor recombinant proteins
JP4469568B2 (en) 2003-07-09 2010-05-26 三菱化学株式会社 Method for producing organic acid
US7875448B2 (en) * 2004-01-12 2011-01-25 Single Use Brx, Llc Bioreactor systems and disposable bioreactor
JP4554277B2 (en) 2004-05-27 2010-09-29 昭和電工株式会社 Method for producing succinic acid by microorganism
DK1773976T4 (en) * 2004-06-04 2020-02-10 Global Life Sciences Solutions Usa Llc SINGLE-BORE ACTOR SYSTEMS AND PROCEDURES
JP4631329B2 (en) * 2004-07-01 2011-02-16 パナソニック株式会社 Angular velocity sensor and manufacturing method thereof
BRPI0707027B1 (en) 2006-02-24 2022-08-16 Toray Industries, Inc METHOD OF MANUFACTURING A CHEMICAL PRODUCT AND CONTINUOUS FERMENTATION APPLIANCE
BRPI0709693A2 (en) * 2006-03-31 2011-07-19 Genencor Int tangential flow filtration apparatus, systems, and processes for the separation of compounds
JP5458481B2 (en) 2007-02-06 2014-04-02 東レ株式会社 Method for producing L-amino acid by continuous fermentation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110832081A (en) * 2017-06-30 2020-02-21 东丽株式会社 Method and apparatus for producing chemical by continuous fermentation
CN110832081B (en) * 2017-06-30 2023-06-27 东丽株式会社 Method and apparatus for producing chemical by continuous fermentation

Also Published As

Publication number Publication date
JPWO2010038613A1 (en) 2012-03-01
BRPI0913137B1 (en) 2021-02-02
CA2738981A1 (en) 2010-04-08
JP5881135B2 (en) 2016-03-09
AU2009299152A1 (en) 2010-04-08
US10428358B2 (en) 2019-10-01
CA2738981C (en) 2018-07-31
US20110177551A1 (en) 2011-07-21
AU2009299152B2 (en) 2015-07-16
BRPI0913137A2 (en) 2020-08-11
PL2330209T3 (en) 2017-12-29
RU2513694C2 (en) 2014-04-20
RU2011117359A (en) 2012-11-10
EP2330209B1 (en) 2017-07-19
US20160168601A1 (en) 2016-06-16
EP2330209A4 (en) 2015-04-01
EP2330209A1 (en) 2011-06-08
CN106011187A (en) 2016-10-12
WO2010038613A1 (en) 2010-04-08
KR20110060888A (en) 2011-06-08

Similar Documents

Publication Publication Date Title
CN102171349A (en) Method for producing chemical and continuous culture system
KR101345160B1 (en) Method of producing chemical product and continuous fermentation apparatus
JP6201753B2 (en) Process for producing chemicals by continuous fermentation and continuous fermentation apparatus
US8551745B2 (en) Method of producing lactic acid by continuous fermentation
JP5978995B2 (en) Process for producing chemicals by continuous fermentation
CN101553572A (en) Method of producing chemical product and continuous fermentation apparatus
JP5659466B2 (en) Method and apparatus for producing chemicals by continuous culture
CN102712936A (en) Method for producing chemicals by continuous fermentation
JP2011188791A (en) Method for operating continuous fermentation apparatus
JP5217736B2 (en) Method for producing D-lactic acid
JP5593594B2 (en) Process for producing chemicals by continuous culture
CN104039970A (en) Method for producing chemical substance

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C12 Rejection of a patent application after its publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20110831